Oncology

Credited Ph.D. programme

Prof. Dr. András Jeney

Apoptotic and proliferative activity in Neuroblastoma and

PNET, tumors of the Thyroid gland and Parathyroid gland

Dr. Parvaneh Farid

Supervisor: Prof. Dr. Béla Szende

Semmelweis University 1st Department of Pathology and Experimental Cancer Research

Budapest, 2003

2

Introduction 3

Apoptosis in general 5

The TUNEL enzymatic labelling assay & immunohistochemistry 9

Morphology of apoptosis 10

Cell Suicide or cell sacrifice 12

Apoptosis in endocrine cells 13

Role of P53 in apoptosis 13

Bcl-2, an inhibitor of apoptosis 16

Function of Bax 17

Retinoic Acid Receptor 18

Neuroblastoma 20

Classification of Neuroblastomas 21

Primitive Neuroectodermal Tumors-PNET 21

Retinoids and apoptosis in Neuroblastoma 22

Genetic heterogeneity of Neuroblastoma 23

Apoptosis and response of Neuroblastoma to chemotherapy 24

Spontaneous apoptosis & RAR incidence in Neuroblastomas & PNET 26

Material & methods 26

Results 29

Discussion 31

Tumors in thyroid glands 33

Apoptosis in Thyroid tumors 35

Materials & methods 36

Results 38

Discussion 39

Our contribution to WHO 42

Tumors of the parathyroid glands 44

Apoptosis in Parathyroid tumors 46

Materials & methods 47

Results 48

Discussion 58

Conclusion 60

Abbreviations 62

References 64

3

Introduction

A noble man of Persia by the name Baha’u’llah (1817-1892) once explained: ”the

stages that mark the wayfarer’s journey from the abode of dust to the heavenly

homeland are said to be seven, the first is the Valley of Search, the steed of this valley is

patience”. This is a traveller’s tale. It has been hoped to tread the path of a real traveller,

passing through the valleys of love, knowledge, unity, contentment, and wonderment,

arriving to the seventh valley, which would lead us to a result in our research.

Over the past two decades there has been an increase in the findings on apoptosis. More

than 30 new molecules have been discovered, whose known functions have exclusively

to do with the initiation or regulation of apoptosis. A further twenty molecules at least,

although already associated with important roles in signalling or DNA replication,

transcription or repair, have been recognised as affecting the regulation of apoptosis.

This remarkable process is responsible for cell death in development, normal tissue

turnover, atrophy induced by endocrine and other stimuli, negative selection in the

immune system, and a substantial proportion of T-cell killing. It also accounts for many

cell deaths following exposure to cytotoxic compounds, hypoxia or viral infection. It is

a major factor in the cell kinetics of tumors, both growing and regressing (1). Many

cancer therapeutic agents exert their effects through initiation of apoptosis, and even the

process of carcinogenesis itself seems sometimes to depend upon a selective, critical

failure of apoptosis that permits the survival of cells after mutagenic DNA damage. It

has been recognized that most if not all physiologic cell death occurs by apoptosis and

that alteration of apoptosis may result in a variety of malignant disorders (2).

Consequently in the last years interest in apoptosis has increased greatly. Great progress

has been made in the understanding of the basic mechanisms of apoptosis and the gene

products involved (3,4).

The aim of our study has been to learn more about the apoptotic and proliferative

activities in certain endocrine cells, namely Thyroid, Parathyroid tumor cells,

Neuroblastoma and some Primary Neuroectodermal Tumors (PNET) as they crossed

our path with their rarity. The value of these parameters was studied regarding the

prognostic and predictive values and their role in differential diagnosis. Having

examined P53, Bcl-2 and Bax and RAR in different tumors of the tissues mentioned

above, new dimension has been observed. Although few apoptotic tumor cells could be

detected in our study in any of the benign or malignant tumors, this may be relevant to

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the high expression of Bcl-2, since Bcl-2 is known to secure cell survival (5). The

expression of Bcl-2, the qualitative and quantitative differences in different tumors of

thyroid glands, be it in the adenomas or follicular adenocarcinomas, compared to its

expression in papillary carcinomas had been observed to vary greatly, worth noticing

while doing differential diagnosis in practice. The level of Bcl-2 expression in tumors

was related to the apoptotic index as measured by morphologic assessments and

terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labelling

(TUNEL) assays of DNA fragmentation (6). The high expression of Bcl-2 in adenomas

of the thyroid gland suggests the susceptibility to transformation to malignancy. With

the knowledge that Bcl-2 inhibits apoptosis in a number of different cells, it becomes

clear that prolonged survival of cells overexpressing Bcl-2 is a factor in predisposition

to malignancy (5). From the practical point of view, determination of P53, Bcl-2 and

Bax ratio in thyroid tumors may contribute to the differentiation between adenomas and

specially follicular carcinomas (7). In parathyroid glands on the other hand the

qualitative or quantitative difference between apoptotic and mitotic activity as well as

P53, Bcl-2 and Bax expression has been examined when parathyroid adenomas and

hyperplasias were compared. Nuclear positivity observed in some of the adenomas may

be the sign of tendency to malignant transformation. Co-expression of P53, Bax and

Bcl-2 has been examined to find a relation between mitotic and apoptotic activity

hoping to find a good reason for the very low rate of malignancy in the parathyroid

glands.

Neuroblastomas are heterogenous in terms of genetic features and clinical behaviour. A

remarkable feature of these tumors is the propensity to undergo spontaneous

differentiation. Furthermore some tumors are very responsive to chemotherapy, but

others are quite resistant. Therefore the spontaneous apoptosis has been examined.

Certain pharmacological agents with variable mechanisms of action can induce

apoptosis in neuroblastoma calls. Of this group the most thorough understanding has

emerged from studies on the retinoids. All-trans retinoic acid (ATRA) can induce

neuroblastoma cells to differentiate or undergo apoptosis depending on the cell

phenotype (8). Several studies have been dedicated to the effects of retinoids on

neuroblastomas. It has been observed that the effects of the retinoids are agent-specific

and dose-dependent (9). 13-cis retinoic acid is capable of lowing growth and inducing

differentiation, at least in vitro. However its effects on inducing programmed cell death

in neuroblastomas are less clear (8). 9-cis retinoic acid is more effective than ATRA at

5

inducing apoptosis in neuroblastoma cells (10). Successful induction of apoptosis by 9-

cis retinoic acid requires that it be cleared from the cells by a washout technique in

culture, which may have implications for in vivo dosing with this agent. In

neuroblastomas the incidence of spontaneous apoptosis as well as its relation to retinoic

acid receptor positivity was examined (11). In cases of PNET, however, the apoptotic

index as well as retinoic acid receptor positivity was quite different from that of

neuroblastoma. The level of Bcl-2 expression in neuroblastomas is inversely related to

the apoptotic index. There have been conflicting reports about the relationship between

Bcl-2 expression and prognostic features associated with this type of tumor when it

comes to the association between Bcl-2 expression and MYCN amplification. The

findings of one group were an association between Bcl-2 expression, Myc N

amplification, and unfavourable histology (12). Other researchers, however, failed to

confirm these correlations (13). In neuroblastoma cell lines, both Bcl-2 and Bcl-Xl (but

not Bcl-Xs) are expressed (14). Bcl-2 is primarily expressed in lines of chromaffin

lineage, Bcl-Xl is expressed in both chromaffin and nonchromaffin lineage

neuroblastoma cell lines (6). Generally neuroblastoma cell lines that express high levels

of one protein express lower level of the other (14).

Apoptosis in general

Like people, cells die in different ways: accident, murder, old age, even suicide. Cells

that are chosen to die either because they are superfluous, diseased, or have served their

useful purpose don’t just fall apart and expire, they go through a predictable, well

choreographed series of events. The cells round up, their outer membranes form bulges

called blebs, nuclear membranes and some internal structures break down, the nuclear

DNA is fragmented by enzymes, and finally the cell breaks into pieces that are

devoured by still vital neighboring cells. This mode of cell death, in which single cells

are deleted in the midst of living tissues, is called ‘apoptosis’. Apoptosis is a well

defined morphological phenomenon. Eventually, the cell breaks into small membrane-

surrounded fragments (apoptotic bodies), which are cleared by phagocytosis without

inciting an inflammatory response. The release of apoptotic bodies is what inspired the

term “ apoptosis “ from the Greek, meaning “to fall away from” and conjuring notions

of the falling of leaves in the autumn from deciduous trees (1).

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Cell death can occur by either of two distinct mechanisms, necrosis or apoptosis

(15,16). In addition, certain chemical compounds and cells are said to be cytotoxic to

the cell, that is, to cause its death. The question here can arise of what’s the difference

between these terms? To clear up these two terminologies, some basic definition will

follow. Necrosis or accidental cell death is the pathological process that occurs when

cells are exposed to a serious physical or chemical insult. Apoptosis (normal or

programmed cell death) is the physiological process by which unwanted or useless cells

are eliminated during development and other normal biological processes. Cytotoxicity

is the cell-killing property of a chemical compound such as food, cosmetic or

pharmaceutical or a mediator cell (cytotoxic T-cell). In contrast to necrosis and

apoptosis, the term cytotoxicity does not indicate a specific cellular death mechanism.

For example, cell mediated cytotoxicity (that is cell death mediated by either cytotoxic

T lymphocytes (CTL) or natural killer (NK) cells combines some aspects of both

necrosis and apoptosis (17,18). There are many observable morphological and

biochemical differences between necrosis and apoptosis (16). Necrosis occurs when

cells are exposed to extreme variance from physiological conditions e.g. hypothermia

and hypoxia which may result in damage to the plasma membrane. Under physiological

condition direct damage to the plasma membrane is evoked by agents like complement

and lytic viruses. Necrosis begins with an impairment of the cell ability to maintain

homeostasis, leading to an influx of water and extracellular ions. Intracellular

organelles, most notably the mitochondria and the entire cell, swell and rupture (cell

lysis). Due to the ultimate breakdown of the plasma membrane, the cytoplasmic content

including lysosomal enzymes are released into the extracellular fluid. Therefore in vivo

necrotic cell death is often associated with extensive tissue damage resulting in an

intense inflammatory response (17). Apoptosis in contrast, is a mode of cell death that

occurs under normal physiological conditions and the cell is an active participant in its

own suicide. It is most often found during normal cell turnover and tissue homeostasis,

embryogenesis, induction and maintenance of immune tolerance, development of the

nervous system and endocrine dependent tissue atrophy. Cells underlying apoptosis

show characteristic morphological and biochemical features (18). These features include

chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm

and nucleus into membrane bound vesicles (apoptotic body) that contain ribosomes,

morphologically intact mitochondria and nuclear material. In vivo, these apoptotic

bodies are rapidly recognized and phagocytized by either macrophages or adjacent

7

epithelial cells (19). Due to this efficient mechanism for the removal of apoptotic cells

in vivo no inflammatory response is elicited. In vitro, the apoptotic bodies as well as the

remaining cell fragments ultimately swell and finally lyse. This terminal phase of in

vitro cell death has been termed secondary necrosis.

Apoptosis or programmed cell death (PCD) is a genetically controlled process whereby

cells die in response to environmental or developmental cues. The morphological

characteristics of apoptosis include cytoplasmic blebbing, chromatin condensation and

nucleosomal fragmentation (2). Apoptosis is relevant to a range of biological processes,

including differentiation, development, cell maturation and immunologic function

(3,20). Dead cells are rapidly phagocytized to prevent damage to neighboring cells. Too

much cell death may produce neurodegenerative diseases and impaired development,

while insufficient cell death can lead to increased susceptibility to cancer and sustained

viral infection. Progress has been made in the past decade to identify many of the basic

components that contribute to apoptosis, including transcriptional mediators, membrane

bound receptors, Bcl-2 family members, kinases/phosphatase, and cysteine proteases.

Bcl-2 was one of the first genes shown to regulate apoptosis (21) and can inhibit

apoptosis in a wide variety of systems. Bcl-2 belongs to a growing family of genes that

can either positively or negatively regulate apoptosis. One of these gene products, Bax,

binds to Bcl-2 and antagonizes its ability to block apoptosis (22). Another critical

element of the apoptotic process is the activation of cystein protease, which is currently

referred to as caspases. In general, the caspases act downstream of Bcl-2-like proteins to

induce apoptosis. Thus the regulation of apoptosis appears to be a precarious balance

between factors that promote survival and those responsible for initiating and executing

cellular suicide or maybe sacrifice.

What causes these morphological changes that we recognize as apoptosis and the

biochemical changes often associated with this phenomenon? The answer is proteases,

especially activation of a family of intercellular cystein proteases which cleave their

substrates at aspartic acid residue, known as caspases for Cysteine Aspartyl-specific

proteases. The observation that caspases cleave their substrates at Asp residues and are

also activated by proteolytic processing at Asp residue makes evident that these

proteases collaborate in proteolytic cascades, whereby caspases activate themselves and

each other (23). Mammalian caspases appear to constitute an autolytic cascade, some

members (notably caspase 8 or FLICE) being “apical” and more susceptible to

modification by endogenous regulatory proteins, while others (notably capase 3 also

8

called CPP32, Yama and apopain) enact the final, irreversible commitment to death.

Study of caspase substrates is providing interesting insights into the ways in which cells

dismantle their structure and function. Such substrates include cytoskeleton proteins

such as actin and fodrin and the nuclear lamins, but also an array of regulatory and

chaperone like proteins whose function is altered by cleavage in subtle and suggestive

ways (24). Caspases appear to be in most if not all cells in inactive proenzyme form,

awaiting activation by cleavage. One of the killing mechanisms of cytotoxic T cells is a

protease, granzyme B, that is delivered to the target cell by the T cell granules and

triggers these latent pro-enzyme. There are endogenous triggers also, and the first to be

discovered, the C. elegans CED4 protein and its mammalian homologue, is particularly

intriguing because of its mitochondrial origin. The CED4 could be the signal that

initiates apoptosis under conditions of shut down of cellular energy metabolism, or

when there is a critical level of cell injury affecting mitochondrial respiration. In this

way CED4 may act as the link between agents long known to be associated with

mitochondrial injury, such as calcium and reactive oxygen species, and the initiation of

apoptosis. A second mitochondrial protein of great significance in apoptosis is BCL-2, a

mammalian homologue of nematode CED9 protein. Bcl-2 has the tertiary structure of a

bacterial pro-forming protein, and inserts into the outer membrane of mitochondria. It

abrogates apoptosis, probably through binding CED4 and another protein Bax, with

which it forms heterodimers and which, like CED4, is also a “ killer ” protein (22). Both

Bcl-2 and Bax have several structurally and functionally similar homologues and some

of this family at least also tap into other cell membranes such as the outer nuclear

membrane and the endoplasmic reticulum.

So are there other sources of death transducers, activating the caspase cascade

because of injury to or signals arising in other parts of the cell than mitochondria? There

are already examples that show that the answer is yes. Thus, the onco-suppressor

protein P53 is activated following some types of DNA damage and can trigger

apoptosis. One way whereby this can happen is through transcriptional activation of

Bax7. The second messenger ceramide, a product of membrane linked acid

sphingomyelinase activation, may act as a signal for plasma membrane damage (25).

And a powerful caspase activating system is mediated by cytokine receptors of the

tumor necrosis factor family, notably fas/apo-1/CD95, TNF receptor I, and others.

These mechanisms for coupling cell injury to apoptosis have mostly depended on

activation of pre-formed proteins. Apoptosis can also be initiated by transcriptional

9

mechanisms, although rather little is known about most of them. An outstanding

example is Drosophila gene reaper, transcriptionally activated around two hours prior to

developemental and injury- induced deaths in this organism. Drosophila apoptosis can

occur without reaper transactivation but requires very substantially enhanced stimuli,

suggesting that reaper adjusts a threshold for apoptosis initiation (26). Another gene

whose transcription can initiate death is the familiar immediate early gene c-myc (27).

Transcriptional activation of c-myc initiates entry into DNA synthesis and is required

for sustained re-entry in repeated cell cycles, but c-myc activation in the absence of

concurrent cytokine support triggers apoptosis. This can also be interpreted as a

“threshold regulatory” effect; c-myc expression increases the cellular requirement for

survival factors such as IGF-1. Impressive confirmation of the significance of these

pathways to apoptosis is available from study of trans forming viruses. These are

hardened survivors in the labyrinth of cell regulation, and have found keys to allow

escape from cell death in a variety of ways. Thus the transforming papovavirus SV40,

adenovirus type 12, Human Papilloma Virus type 16 and Epstien-Barr Virus all have

proteins that inactivate apoptois through inactivation of P53 or binding of Bax (28).

There are transcriptional and non-transcriptional pathways for activation of

apoptosis, and they play through common effector events mediated by caspases and

regulated by members of the Bcl-2 family. Underlying this simple scheme, however, is

an extraordinary complexity. Thus inactivation of Fas signalling appears to neutralize

the ability of both c-myc and P53 to initiate apoptosis (29). Many of the proteins

mentioned above have alternative splice variants that have apposite effect. And we still

have little idea of the relevance of intracellular location or of the cell lineage to the

activity of most of the apoptosis proteins. Many other gene products, including

oncoproteins such as RAS and ABL, can influence susceptibility to apoptosis but in

some cases a single oncoprotein may either increase or decrease susceptibility

depending on the context. Perhaps it is not surprising that a cellular function as

important and irreversible as death should be subject to a huge range of course and fine

controls.

The TUNEL enzymatic labelling assay

One of the methods for studying apoptosis in individual cells

There are different methods by which apoptosis could be detected in individual cells,

enzymatic labelling and staining with fluorochrome. In our study we have used

10

enzymatic labelling, the so-called TUNEL enzymatic labelling assay. Extensive DNA

degradation is a characteristic event, which often occurs, in the early stage of apoptosis.

Cleavage of the DNA may yield double-stranded, LMW DNA fragments (mono-and

oligonucleosomes) as well as single strand breaks (nicks) in HMW-DNA. Those DNA

strand breaks can be detected by enzymatic labelling of the free 3’-OH termini with

modified nucleotides (X-dUTP, X= biotin, DIG or fluorescein). Suitable labelling

enzymes include DNA polymerase (nick translation) and terminal deoxynucleotidyl

transferase (end labelling). DNA polymerase I catalyze the template dependent addition

of nucleotides when one strand of a double-stranded DNA molecule is nicked.

Theoretically, this reaction (In Situ Nick Translation, ISNT) should detect not only

apoptotic DNA, but also the random fragmentation of DNA by multiple endonucleases

occurring in cellular necrosis. Terminal deoxynucleotidyl transferase (TdT) is able to

label blunt ends of double-stranded DNA break independent of a template. The end-

labeling method has also been termed TUNEL (TdT-mediated X-dUTP nick end

labelling). The TUNEL method is more sensitive and faster than the ISNT method. In

addition, in early stages cells undergoing apoptosis were preferentially labelled by the

TUNEL reaction, whereas necrotic cells were also identified by in situ nick translation

(ISNT). The in situ nick translation (ISNT) is template dependent and TUNEL is

template independent; experiments suggest the TUNEL reaction is more specific for

apoptosis and the combined use of the TUNEL and ISNT is helpful to differentiate

cellular apoptosis and necrosis (30).

Morphology of apoptosis Apoptosis may be divided into three distinct stages: Commitment, execution and

clearance. It is becoming increasingly apparent that a variety of factors influence a cell’s

commitment to the death program in response to an apoptotic stimulus. These include

the cell’s metabolic state, the extent and type of damage induced by the agent, the cell’s

genotype, and the relative expression of growth,- survival-, and death-promoting

factors. Disturbances in any one of these factors may affect a cell’s susceptibility to

apoptotic stimuli, resulting in an inappropriate cellular fate, possibly having significant

consequences on tissue development or disease progression. Altered susceptibility to

apoptosis is particularly relevant to tumorigenesis and tumor progression and for the

development of effective cancer treatment strategies. In the stage of commitment the

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cell having received a potentially lethal apoptotic stimulus, becomes irreversibly

commited to death. It is in the execution stage where major structural changes occur.

Clearance is when cellular remnants are removed by phagocytosis. The structural

changes that occur during the execution phase were described by Kerr et al (1) and are

now becoming better understood mechanistically. During the execution phase,

coordinated morphological and biochemical changes occur within the nucleus,

cytoplasm, organelles, and plasma membrane. The most easily recognizable features of

these events are changes that occur within the nucleus. Chromatin condenses and

aggregates along the nuclear periphery in a crescent shaped pattern. Molecular

characterisation of the chromatin reveals an ordered degradation of the DNA by a

cation-dependent endogenous nuclease, first into large fragments and finally into

nucleosomal fragments. However, cleavage of chromatin to nucleosomal fragments

does not occur in all cell types and can be inhibited without blocking the other changes

of apoptosis (31,32). Parallel with chromatin condensation, the nuclear ultrastructure is

changed. The nuclear lamina, an intermediate filament network that maintains nuclear

envelope integrity and nuclear pore distribution, is proteolytically cleaved (33,34).

Disruption of the nuclear structural framework may then allow nuclear pore clustering

(35) and fragmentation of the nucleus into chromatin-containing fragments, many of

which retain a trace of the nuclear membrane. Other cytoplasmic organelles remain

structurally intact, although mitochondrial dysfunction is associated with apoptosis (36).

Reduction in the mitochondrial transmembrane potential, uncoupling of electron

transport from ATP synthesis, and increased generation of reactive oxygen species

precede the nuclear changes. In the cytoplasm, protein cross- linking occurs through the

action of transglutaminase (37), cytoskeletal filaments aggregate in parallel arrays, and

the endoplasmic reticulum dilates and fuses with the plasma membrane, creating pock-

like craters at the point of fusion. The structural integrity of the plasma membrane is

further compromised by the loss of membrane phospholipid asymmetry, microvilli, and

cell-cell junctions. The cell rounds up, dissociates itself from its neighbours, and shrinks

dramatically, throwing out protuberances that separate into membrane bound ‘apoptotic

bodies’. Within tissues, apoptotic cells and apoptotic bodies are recognized and rapidly

phagocytosed by neighboring cells or macrophages. Once the execution phase of

apoptosis is activated, the entire process proceeds rapidly, and is completed within a

few hours (38). The duration of this phase is relatively invariant with respect to cell type

and apoptotic stimulus, suggesting that the final stage of apoptosis proceed through a

12

common pathway. However, the commitment phase, the time from reception of the

apoptotic stimulus until irreversible initiation of the execution phase is extremely

variable, being dependent on cell type, apoptotic stimulus position within the cell cycle,

and expression of various death modulating factors. Many of the factors that influence

commitment or cellular susceptibility to apoptosis, are involved directly in the reception

and transduction of the apoptotic signal. Strikingly most of the genes involved in the

regulation of apoptosis that have been identified during the past few years have turned

out to be also of importance in carcinogenesis.

Cell suicide or cell sacrifice

Apoptosis, or programmed cell death, is a critical process in many areas of biology such

as morphological modelling of tissues during development, removal of autoreactive

immune cells, hormonal or age-related tissue atrophy and normal cell turnover. During

apoptosis, cells, by virtue of their own genetic makeup, die and are deleted for the

overall good of the organism (39). It is also said in literature that cells in multicellular

organisms can kill themselves by activating a suicidal genetic program in response to a

wide variety of signals, including hormones, cytokines, ionising radiation, and

chemotherapeutic agents and that this process of cell suicide is called apoptosis which

usually occurs under physiological conditions (40). The phenomenon of apoptosis has

long been known as programmed cell death, which is fundamental for embryonic

development, such as in metamorphosis, morphogenesis and synaptogenesis (41). While

apoptosis is energy requiring process, functional mitochondria are necessary to provide

adequate energy for the process to reach completion, so it might not be quite fair to call

the process a suicidal process, with a negative connotation of lacking energy and will

power. If cells die because of a signal, be it cytokines or chemotherapy, then they have

been killed and murdered. On the other hand if there is a process where cells die by

virtue of their own genetic make up for the overall good of the organism then this is a

sacrifice a cell makes, since sacrifice involves renouncing that which is lower for that

which is higher. This happens during the embryological development, when a cell

which can not serve the purpose anymore dies and is deleted from the environment. A

tree which bears no fruit is good for fire. It is worthwhile reviewing the terminologies

that have been given to certain processes in the life of a cell.

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Apoptosis in endocrine cells

Because of the fine balance that is maintained between cell proliferation and

death, there is little change in the overall number of cells during the adult life of

multicellular organisms. Cell generation occurs via mitosis, a process that is relatively

well understood compared to the process by which cell die. With regard to cell death,

there are two distinct mechanisms by which this process can occur: necrosis and

apoptosis. Necrotic cell death is characterized by cell swelling and lysis, with the cell

playing no active part in the process. It is also associated with a characteristic

inflammatory response. Apoptosis, in contrast, is a genetically controlled process and is

probably the counterbalancing force to mitosis under normal physiological conditions.

In contrast to mitosis, very little is known about how apoptosis is regulated, but it is

known that the cell plays an active part in its own demise. A variety of genes that play

regulatory role in apoptosis have been identified. These fall into two distinct groups,

those that drive the process and those that inhibit it. In the former category are genes

such as myc and P53 and in the latter genes such as Bcl-2 and Bcr-abl. What is striking

about this observation is that many of them are oncogenes and have been implicated in

the development of numerous cancers. Bcl-2, Bax and P53 expression has been

examined by us (42) in different endocrine cells as factors influencing apoptosis by

either suppressing, inhibiting or causing apoptosis. Our study has been aiming to help

the differential diagnosis in the case of thyroid and parathyroid tumors and in giving

weigh to predictive values. It is important to ponder upon the role and function of these

proteins, finding out how and in what way they act in the process.

Role of P53 in apoptosis

The tumor suppressor gene P53 is the most commonly mutant gene in human cancers

(43). P53 protein levels rise in response to genotoxic damage. This rise in p53, which

results from increased transcription and protein stabilization, is responsible for initiating

G1/S-phase cell cycle arrest, DNA repair and /or apoptosis. Because p53 is a principal

mediator of the cellular response to genotoxic damage, its altered expression and

function may allow the propagation of potentially harmful genetic lesions leading to

oncogenesis. The first clear demonstration that p53 expression could induce apoptosis

14

was based on a cultured cell line transfected with a temprature-sensitive p53 mutant

(44). P53 can induce apoptosis in different ways 1)by activating bax, a proapoptotic

member of bcl-2 family, 2) by enhancing the expression of killer receptor, 3) by

preventing such genes transcription which their products inhibit p53-dependent

apoptosis, 4) by protein-protein mutual effect e.g. it binds to transcription/ repair

complex (45). In 1993 concurrent studies by Lowe et al (46) and Clarcke et al (47)

demonstrated that p53 was involved in the cellular apoptotic response to genotoxic

damage. In these studies, thymocytes from mice rendered homozygous null for p53 by

germline targeting were found to be completely resistant to the apoptosis seen in wild

type cells shortly after exposure to ionising radiation or chemical induced DNA double-

strand breaks (46,47). However, p53 genotype had no effect on glucocorticoid- induced

apoptosis in these cells. These findings established the existence of distinct p53-

dependent and p53 independent mechanisms of apoptosis. Moreover, because p53-null

mice showed a high incidence of thymic lymphomas, the results gave some support to

the hypothesis that p53 mutations may facilitate tumor initiation and progression by

producing defects in apoptosis. This hypothesis has been further supported by elegant in

vivo experiments using transgenic mice engineered to selectively express SV40 large T

antigen product, which can selectively inactivate Rb or p53, in choroid plexus cells. In

the animals expressing the full length SV40 large T transgene, which inactivates both

Rb and p53, aggressive choroid plexus tumor formation was observed. In contrast,

tumor growth occurred at a considerably slower rate in animals expressing a truncated

SV40 large T transgene capable of inactivating Rb but not p53 (48). Analysis of these

tumors revealed that p53 genotype had no appreciable effect on rates of proliferation.

Instead, rates of apoptosis were low in those tumors in which p53 was inactivated,

suggesting that p53 associated tumor suppression due to enhanced rate of apoptosis

rather than decreased rate of proliferation. Similar results have been reported in

additional tissue-specific transgenic system that addressed the role of p53 and Rb in

tumor development (49,50).

Gene expression has been shown to be required for both apoptosis and survival

depending on the cell type and stimulus. Indeed, inhibition of RNA and protein

synthesis can block apoptosis induced by a number of circumstances. Others have

shown that in some cases RNA and protein synthesis inhibitors either do not block or

actually promote cellular demise (51). The basic machinery for apoptosis is

constitutively expressed but can be modulated by changes in Gene expression in order

15

to trigger the death program. p53 was first detected in rodent cells transformed by

simian virus SV40 in a complex with the transforming protein SV40 T antigen,

suggesting that it plays a role in growth control (52). Subsequently, it was noted that

p53 mutations occur in a wide variety of tumors including lung, breast, colon,

esophagus, liver, bladder, ovary, brain and haemopoetic tissues (43). In fact, p53 loss-

of- function mutations are the most common genetic alteration found in human cancer.

Furthermore, overexpression of wild-type p53 can suppress tumor formation in culture

(53,54,55). These data suggest that p53 function as a tumor suppressor gene and has

sparked extensive research into understanding its mechanism of action. p53- mediated

transrepression, on the other hand, occurs on genes lacking the p53 DNA binding site

and is probably dependent on interactions with components of the basal transcriptional

machinery (56,57). Mutational analysis of p53 has revealed distinct domains that

contribute to its gene regulatory activity. Consistent with its role as a tumor suppressor

gene, expression of p53 can induce either cell cycle arrest in G1 or apoptosis (58,44).

Functional p53 is required for apoptosis induced by ionizing radiation and

chemotherapeutic drugs (59,47) as well as transforming oncogenes like c-myc (60,61).

The function of p53 is likely to depend on the cell type and/or physiological

circumstances. Indeed, cytokines (44) and growth factors can affect the ability of p53 to

induce apoptosis or growth arrest. These results have led to the hypothesis that

genotoxic induction of p53 acts as a checkpoint control to stop further progression in the

cell cycle and thereby maintain genomic integrity (62). The mechanism by which p53

induces apoptosis, however, is somewhat unclear. Generally, p53 mediated apoptosis is

dependent on its gene regularity capability. Another critical issue that remains is

whether p53-dependent cell death involves activation or repression of cellular genes.

Studies have indicated that at least in some cases p53 induced apoptosis is not affected

by the presence of RNA and protein synthesis inhibitors, suggesting that repression

rather than activation is the primary mechanism (63). Several p53 responsible genes

have been identified that could affect cell survival, including bcl-2 (64). Concerning

thyroid neoplasms there exists various reports on the overexpression of p53 especially

in poorly and undifferentiated carcinomas (65,66).

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Bcl-2, an inhibitor of apoptosis

One of the first mammalian genes discovered to regulate cell death was the anti-

apoptotic gene bcl-2 (67). The bcl-2 proto-oncogene was originally discovered as a

common translocation in non-Hodgkins B-cell lymphoma (68,69). This chromosomal

translocation event places the bcl-2 gene under the transcriptional control of the

powerful enhancer elements of the immunoglobulin heavy chain resulting in high levels

of bcl-2 expression and the abrogation of normal programmed cell death and promotion

of malignant potential. Disruption of bcl-2 in “ knock out “ mice leads to impaired

kidney function manifested by polycystic disease and postnatal immune failure due to

dramatic cell loss through apoptosis. Thus, gain of bcl-2 function is associated with

tumor development (68,69,70,71,72), while loss of bcl-2 has only restricted

consequences to normal development. This suggests that there may be some redundancy

in the bcl-2 family or that other members have a more critical role. Several bcl-2 like

genes have been identified that can promote, rather than inhibit, apoptosis. The best-

characterized and prototypic gene of this class is bax (22). While overexpression of bax

can induce apoptosis, disruption of this gene in “ knock out “ mice leads to lymphoid

hyperplasia (73). Recent data also demonstrates that expression of bax can suppress

tumorigenesis in vivo (74). These data suggest that bax plays a critical role during

apoptosis. Biochemical studies have indicated that bax directly interacts with itself as

well as several anti-apoptotic proteins such as bcl-2 (22). Interaction between death

promoting and suppressing bcl-2- like proteins has lead to a model for explaining how

these proteins function. According to this model, the ratio between survival factors,

such as bcl-2, and death promoters, such as bax, controls the fate of the cell. However,

the biochemical mechanism of these proteins is still unclear. What is also unclear is

which set of bcl-2 proteins act as effector molecules. For example, do the death

promoting genes simply inhibit a required survival factor (e.g. bcl-2, bcl-xl ) or do they

actually trigger apoptosis?

Bcl-2 and its family members bind to a number of unrelated proteins that has provided

insight into their apoptotic activity. Indeed, bcl-2 related proteins are generally found

associated with membrane structures, particularly the mitochondria, endoplasmic

reticulum, and the nuclear envelope. Furthermore, in vitro experiments demonstrate that

bcl-xl can facilitate ion transport across a lipid bilayer. It will be of interest to determine

if the bcl-2 family members actually function as channels in vivo and if this activity is

17

responsible for apoptosis regulation. These and other studies have lead to several

hypotheses regarding the biochemical mechanism of this family of proteins. One

possibility that has emerged is that they form a channel capable of regulating ion flux,

such as calcium. Calcium is a critical component of signaling pathways and high level

of intracellular calcium is often associated with cell death, bcl-2 can suppress calcium

release from the endoplasmic reticulum, although it is possible that this is simply a

downstream consequence of its ability to inhibit apoptosis (75). Nevertheless, the

experiments indicating that the bcl-2- like proteins can form ion channels add some

credence to this model. Others have speculated that bcl-2 regulates the generation of

reactive oxygen radicals since bcl-2 is able to attenuate cell death induced by oxidative

damaging agents. (75). Free radicals can contribute to cell death by disrupting DNA,

proteins, and lipids. The localization of bcl-2 at mitochondrial membranes makes it

plausible that the protein is directly involved in oxidative phosphorylation (67).

However further evidence demonstrates that bcl-2 can inhibit apoptosis in cells that lack

mitochondrial DNA and thereby respiration (76). An alternative mechanism for bcl-2

action at the mitochondria may be the control of cctochrom C release. Release of

cytochrome c from the mitochondria into the cytosol is required for caspase activation

and DNA fragmentation in cell free extracts and bcl-2 can act to suppress cytochrom c

efflux from mitochondria (77). It will certainly be important to determine if efflux of

proteins, such as cytochrom C, from the mitochondria actually contributes to apoptosis

in vivo or if this is simply a downstream phenomenon. Additionally, there is tremendous

interest in elucidating the down stream events of apoptosis, most notably caspase

activation.

As to the expression of bcl-2 in endocrine cells, bcl-2 has been reported to be retained in

well & poorly differentiated thyroid carcinomas but absent in undifferentiated thyroid

carcinomas (159,41). Several studies have found that bcl-2 is widely expressed in

neuroblastoma tumors and tumor derived cell lines (13,54).

Function of Bax

Bax has been shown by a number of investigators to be capable of forming either

homodimers with bcl-2 and bcl-Xl by immunoprecipitation and yeast two hybrid select

systems (40,41). Bases on the tendency of bax to promote cell death, it has been

proposed that the relative ratio of bax heterodimers to bax homodimers serves as a

18

switch that dictates the cell fate (78,79). According to this model, exposure of the cell to

cytotoxic insults may promote the formation of bax homodimers. However this death

initiation step may be blocked, if sufficient quantities of the prosurvival factors bcl-2 or

bcl-Xl are present to form heterodimers with bax. Thus depending on the relative ratio

of bax heterodimers to bax homodimers, the cell may proceed toward either cell

survival or cell death. bax dependent cell killing appears to be linked to caspase

activation (80,81). Several reports have suggested that overexpression of bax leads to

the loss of inner mitochondrial potential, a phenomenon also known as mitochondrial

permeability transition (82,83). Observations taken together have led to the proposal

that bax may form pores on mitochondrial surface, leading to the dissipation of

mitochondrial potential and to the release of cytochrome C (84). One could envision

that these series of processes would first require a signalling mechanism that switches

bax from its normal physiologically inactive state, possibly represented by its cytosolic

form, to an active apoptotic state possibly represented by its mitochondria-bound form.

Studying the expression of bax has not been given too much attention in endocrine cells.

Nevertheless there have been studies that bax expression has been negative in normal

thyroid tissue and weakly positive in meudullary thyroid tumor (85).

Retinoic Acid Receptor

Retinoids have profound effects on normal vertebrate development and the maintenance

of normal cellular functions in some adult tissues (86). A group of pharmacological

agents with different mechanism of action can induce apoptosis in neuroblastoma cells

(8). Some examples are retinoids. All-trans retinoic acid (ATRA) can induce

nueroblastic cells to undergo apoptosis. It is therefore not out of place to expand on

Retinoic Acid Receptor (RAR) since we have examined its expression in

neuroblastomas and PNET and its relation to apoptosis (11).

The widespread cellular effects of retinoids are mediated by two classes of nuclear

receptor: the retinoic acid receptors (RAR) and the retinoid X receptors (RXR) (87).

Discovery of the nuclear retinoic acid receptors (RARs) and nuclear retinoid X

receptors (RXRs), has significantly advanced our understanding of the biological basis

of retinoic acid (RA) function. These receptors, which belong to the multigene family of

steroid/thyroid hormone receptors RA-inducible transcriptional regulatory factors

transduce the RA signal at the level of gene expression. There are 3 subtypes (? , ? and

19

? ) of both RARs, and RXRs, each coded for by separate genes. Nuclear retinoid

receptors are ligand-dependent transcription factors which bind specific DNA

sequences, known as retinoic acid response elements (RARE) in the promoter regions of

target genes. RAR-RXR heterodimers, rather than homodimers of individual receptors,

bind with greatest affinity in vitro to RAREs. Single and combination gene knockout

studies in mice, for each of the nuclear retinoid receptors, have demonstrated that

specific receptor subtypes have signaling roles in certain embryonal tissues, however,

functional redundancy also exists between receptor subtypes in other tissues (88). A

large number of tumor cell lines from different tissue origins undergo growth arrest and,

in some cases, differentiation, following retinoid treatment in vitro (88). These

observations have raised the possibility that disordered retinoid signaling may

contribute to some tumorigenic processes, and, moreover, that retinoids may have

therapeutic or chemo-preventive roles in certain human cancers. Abnormal function or

reduced expression of specific RAR, but not RXR, subtypes contribute to the malignant

phenotype in several human cancers (89,90,91,92,93). Several groups have linked

reduced RAR ? expression to the genesis of human epidermal, lung, and breast cancer

(91,93,94,95).

20

Tumors investigated in our study

1. Neuroblastoma

The neuroblastic tumours, derived from primordial neural crest cells which

ultimately populate the sympathetic ganglia, adrenal medulla and other sites, (96) are an

enigmatic group of neoplasms which have the highest rate of spontaneous regression of

all human malignant neoplasms yet one of the poorest outcomes when occuring as

disseminated disease in children. Neuroblastoma is one of the most common childhood

extracranial solid tumors. Alone, it accounts for about 15% of all childhood cancer

death (97). A great deal is known about the molecular biology and genetics of this

neoplasm. Studies strongly suggest that there are at least two, possibly three,

clinicobiologic subsets of neuroblastoma, ranging from those that rarely kill, however

hopeless the clinical situation may appear to be, to those that rapidly cause death despite

all therapeutic efforts. Furthermore, some tumors are very responsive to chemotherapy

but others are quite resistant. Thirty-five percent of these tumors appear during the first

year of life, 20% during the second year. Altogether, 85 to 90% are found in children

younger than five years of age. Most occur sporadically, but in about 20% of instances

there is strong evidence of a hereditary predisposition.

In childhood, about 25 to 35% of neuroblastomas arise in the adrenal medulla. The

remainder occur anywhere along the sympathetic chain, with the second most common

location being the paravertebral region of the posterior mediastinum. Closely following

is the paravertebral region in the lower abdomen, but tumors may arise in numerous

other sites, including the pelvis and neck and within the brain. By contrast, rare

neuroblastomas in adults are found in the head, neck, and legs. Macroscopically,

neuroblastomas range in size from minute nodules (the “ in situ lesions “) to large

masses more than 1 kg in weight. In situ neuroblastomas are reported to be 40 times

more frequent than overt tumors. The great preponderance of these lesions

spontaneously regress leaving only a focus of fibrosis or calcification in the adult.

Indeed, some clinically overt neuroblastomas have regressed or, alternatively, have

undergone differentiation and maturation into a relatively benign ganglioneuroma.

Some neuroblastomas are sharply demarcated and may appear encapsulated, but others

are far more infiltrative and invade surrounding structures, such as the kidneys, renal

vein, and vena cava, and envelope the aorta.

21

Histologically, there is a considerable range of differentiation among these neoplasms.

Most are composed of small, primitive-appearing cells with dark nuclei, scant

cytoplasm, and poorly defined cell borders growing in solid sheets.

Classification of Neuroblastomas

The International Neuroblastoma Pathology Committee (INPC) has adopted a

prognostic system modeled on one proposed by Shimada et al(98). It is an age- linked

classification dependent on the differentiation grade of the neuroblasts, their cellular

turnover index, and the presence or absence of Schwanian stromal development. Based

on morphologic criteria defined in this article, neuroblastic tumors (NTs) were classified

into four categories and their subtypes:

1. neuroblastoma (Schwannian stroma-poor), undifferentiated, poorly

differentiated, and differentiating;

2. ganglioneuroblastoma, intermixed (Schwannian stroma-rich);

3. ganglioneuroma ( Schwannian stroma-dominant ) maturing and mature;

4. ganglioneuroblastoma, nodular ( composite Schwannian stroma-rich / stroma-

dominant and stroma-poor).

1/a. Primitive Neuroectodermal Tumors (PNET)

Some tumors, although of neuroectodermal origin, express few, if any, of the

phenotypic markers of mature cells of the nervous system and are described as poorly

differentiated, or embryonal, meaning that they retain cellular features of primitive,

undifferentiated cells. The most common is the medulloblastoma, which accounts for

20% of the brain tumors of childhood and occurs in the cerebellum. Other poorly

differentiated tumors occur in other locations, and have been collectively termed

primitive neuroectodermal tumors (PNET). Most classification systems continue to

recognize the medulloblastoma as a distinct clinicopathologic category and

acknowledge the presence of other poorly differentiated tumors that occur

predominantly in children. Primitive neuroectodermal tumor is a highly controversial

designation, which has been applied to neoplasms, primarily of childhood, that are

histologically similar to medullobastoma but occur in locations other than the

22

cerebellum, especially the cerebral hemispheres. Many would prefer not to gather all

these poorly differentiated tumors into a single category until there is a clearer vision of

their biological behavior; others find the designa tion useful to evaluate the efficacy of

chemotherapeutic protocols. More information is needed to settle this issue.

Retinoids and Apoptosis in Neuroblastoma

A group of pharmacological agents with variable mechanisms of action can induce

apoptosis in neuroblastoma cells in vitro. Some examples are retinoids, DNA strand

breaking agents like cisplatin and doxorubicin, protein kinase inhibitors and others. Of

this group the most thorough understanding has emerged from studies on the retinoids.

All-trans retinoic acid (ATRA) can induce neuroblastoma cells to differentiate or

undergo apoptosis, depending on the cell phenotype (8). Three phenotypic variants of

neuroblastoma cells in culture have been described. N-type cells constitute the

neuroblastic phenotype. These cells have neurotic processes and display biochemical

characteristic of neuronal cells. S-type cells are substrate-adherent, epithelial in

phenotype, and display biochemical features of immature Schwann, glial, or

melanocytic cells. The third phenotypic variant is I-type cells, with intermediate

morphology and bio-chemical features of both N-and S-type cells. S-type cells do not

express Bcl-2 and are particularly sensitive to induction of apoptosis by ATRA. This is

in contrast to N-type cells, which express Bcl-2 and are less sensitive to RA-induced

apoptosis (99,100). Neuroblastoma cells that differentiate in response to RA are

resistant to chemotherapy- induced apoptosis. This resistence is associated with

regulation of Bcl-2(100). The effect of the retinoids are agent specific and dose-

dependent. 13-cis RA is capable of slowing growth and inducing differentiation, at last

in vitro. However its effect on inducing programmed cell death (PCD) in

neuroblastomas are less clear (8,9). 9-cis RA is more effective than ATRA at inducing

apoptosis in neuroblastoma cell (10). Successful induction of apoptosis by 9-cis RA

requires that it be cleared from the cells by a washout technique in culture, which may

have implications for in vivo dosing with this agent (10). Finally there is a novel

retinoid called N-(4-hydroxyphenyl) retinamide (fenretinide), which appears to be

particularly potent at inducing apoptosis (101,102). The mechanism through which the

retinoids induce apoptosis has not been clearly defined.

23

Neuroblastoma is an embryonal childhood tumor of neural crest origin with a

unique capacity for spontaneous regression and differentiation, in vivo (103,104,105).

In some older patients the tumor may differentiate into a benign ganglioneuroma.

Unfortunately in the majority of patients, neuroblastomas are metastatic at diagnosis and

most of these children ultimately die of their disease, especially if they are over 1 year

of age at diagnosis (96).

Several studies have found that Bcl-2 is widely expressed in neuroblastoma tumors and

tumor derived cell lines (13). Moreover the level of Bcl-2 expression in tumors is

inversely related to the apoptotic index, as measured by morphologic assessments and

terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end- labelling

(TUNEL) assays of DNA fragmentation (6). There have been conflicting reports about

the relationship between Bcl-2 expression and prognostic features associated with this

disease. One group reported an association between Bcl-2 expression, MycN

amplification, and unfavourable histology (84). Other investigations however have

failed to confirm these correlations (13). In neuroblastoma cell lines, both Bcl-2 and

Bcl-Xl (but not Bcl-Xs) are expressed. Bcl-2 is primarily expressed in lines of

chromaffin lineage, Bcl-Xl is expressed in both chromaffin and nonchromaffin lineage

neuroblastoma cell lines. Generally neuroblastoma cell lines that express high levels of

one protein express lower levels of the other (6).

Retinoid treatment of most neuroblastoma tumor cell lines induces growth arrest and

neuritic differentiation in vitro (106) and, thus provides an excellent model for studying

whether abnormal retinoid signaling is a component of the neuroblastoma tumorigenic

process. Untreated neuroblastoma cells express high levels of RAR-? , low level of

RAR-?, and barely detectable levels of RAR ? , while retinoid treatment induces RAR-

? expression some 40-fold and RAR ? expression by four-fold (107,108,109).

Comparative studies of neuroblastoma cells transfected with each of the 3 RAR subtype

have shown that only RAR-? transfectants demonstrates enhanced retinoid sensitivity

and growth inhibition in the absence of added retinoid (110).

Genetic heterogeneity of neuroblastoma Several genetic features have been found to be characteristic of subsets of

neuroblastomas, including hyperdiploidy with whole chromosome gains, amplification

of the MYCN oncogene, and deletions of the short arm of chromosome 1 (1p)(111).

24

Hyperdiploidity is characteristic of tumors in infants, particularly those who are prone

to undergo spontaneous regression or who respond well to chemotherapy. However, the

most aggressive tumors particularly those who are metastatic in older patients

frequently have MycN amplification and the majority of these also have 1p deletion

(111). Partial trisomy for 17q is also a common genetic change in neuroblastomas and

its presence in the karyotype is generally associated with a worse outcome. Other

deletion and rearrangements are also found such as deletion of 11q or 14q, but so far

these features have not been consistently associated with clinical behaviour.

Apoptosis and response of neuroblastoma to chemotherapy

A variety of DNA damaging agents have been shown to induce apoptosis in

neuroblastoma cells in vitro (112,113). Several stud ies have indicated that

neuroblastoma cells undergo apoptosis in response to DNA damaging agent after arrest

in G2/M of the cell cycle (114). Neuroblastoma cells treated with these agents show

classic morphologic features of apoptosis and evidence of DNA damage. The CD95

system has recently been shown to mediate apoptosis induced by chemotherapy in

neuroblastoma (115). CD95, a member of the TNFR superfamily, triggers cell death in a

variety of cell types. In neuroblastoma cells, doxorubicin, cisplatin, and etoposide

induce expression of CD95, as well as the ligand, CD95L (115). Chemotherapy induced

apoptosis mediated by CD95 in neuroblastoma involves activation of ced-3-like

proteases, resulting in cleavage of PARP an enzyme involved in DNA repair (115).

These results suggest that autocrine or paracrine activation of CD95/CD95L leads to a

death response in neuroblastoma cells after treatment with these specific

chemotherapeutic drugs.

Both Bc-2 and Bcl-Xl can modulate chemotherapy induced apoptosis in neuroblastoma

cells. Single-gene transfection studies have shown that deregulated expression of Bcl-2

or Bcl-Xl in a dose dependent manner can confer resistance to apoptosis induced by

cisplatin or 4-hydroxycyclophosphamide, and delay apoptosis induced by etoposide

(116,112,113).

Not all cytotoxic agents induce apoptosis by engaging CD95. Betulinic acid (BA), a

pentacyclic triterpene, induces apoptosis in a number of cell types, including

neuroblastoma, and this appears to be independent of the Cd95 and P53 pathways (117).

BA-induced apoptosis is associated with increased caspase activity, enzymatic

25

processing of the upstream caspase component, FLICE (caspase-8) and PARP cleavage.

BA-induced apoptosis, however, is independent of CD95/CD95L activation. BA-

induced apoptosis is associated with induction of the apoptosis- facilitating proteins, Bax

and Bcl-Xs. Both Bcl-2 and Bcl-Xl can inhibit apoptosis induced by BA in a manner

similar to its protection from DNA-damaging agents (115).

Caspase activation appears to be the final common pathway for induction of apoptosis

by most agents. Staurosporin, an alkaloid inhibitor of protein kinases, is also able to

induce apoptosis in neuroblastoma cells (118). Staurosporin- induced apoptosis is

associated with proteolytic processing of CPP32 (caspase-3), PARP activation, and

PARP cleavage. In some systems, however, PARP activation is not always associated

with proteolytic processing of PARP. Peroxynitrate (PN), which forms through a

reaction of superoxide with nitrous oxide has recently been shown to induce apoptosis

in the NSC34 neuroblastoma spinal cord cell line (119). In this system, PN-induced

apoptosis was associated with morphologic features of apoptosis and increased PARP

immunoreactivity, but there was no evidence of PARP cleavage. It has been suggested

that PN induce DNA damage that stimulates PARP, which depletes intracellular energy

stores, resulting in cell death (119). This possibility will require further investigation,

however, these results suggest that additional pathways may function to mediate

specific drug- induced apoptosis in neuroblastoma.

Therapeutic strategies based on in vitro studies of neuroblastoma cells have led to

several current clinical trials in patients with neuroblastoma. Most notable are the

protocols utilizing retinoic acid (RA) in the treatment of advanced stage disease, which

are based on in vitro assays indicating RA can inhibit neuroblastoma proliferation. A

phase 1 trial utilizing 13 cis-RA following bone marrow transplantation for stage 4

neuroblastoma has suggested that the drug is well tolerated, with minimal toxicity (120),

and its therapeutic efficacy is currently under investigation.

An improved understanding of the molecular pathways controlling the survival/death

response of neuroblastoma cells should provide insight into mechanism that could

ultimately be targeted for novel therapeutic intervention. In support of this possibility

are studies indicating that neuroblastoma cells, regardless of their Bcl-2 or Bcl-Xl

expression, are sensitive to induction of apoptosis by adenoviral-mediated expression of

the death-facilitating protein Bcl-Xs (116). The possibility of therapeutically engaging

the death pathways directly is intriguing, but will require the development of targeted

gene therapy approaches. Finally, therapeutic interventions aimed at either blocking the

26

Trk ligand-receptor interaction, or selectively inhibiting the Trk family of receptors,

may provide a therapeutic approach that is more effective and less toxic than currently

used chemotherapeutic agents. The blue letters are my own work.

Our studies on apoptosis in Neuroblastoma & PNET

Aim: RAR-ß and apoptosis: therapy with RA

The specimen has been received from the 2nd Department of Pediatrics, Semmelweis

University of Medicine, Budapest, Hungary. The great help and assistance of Prof.Dr.

Dezso Schuler and the co-operation of Dr. Peter Hauser and Dr. Maria Babosa is deeply

appreciated.

Little is known about the spontaneous apoptotic activity in neuroblastomas and PNET.

These tumors have a poor prognosis even when treated with irradiation and/or cytotoxic

drugs (121,122). Recently retinoic acid has been reported to be effective in the therapy

of NB, promoting the differentiation of the tumor cells (123). In vitro studies of NB

cells showed that retinoic acid administration induces apoptosis in cell cultures and this

effect was mediated by a retinoic acid receptor (124). The possibility of inducing

apoptosis by retinoic acid treatment has been studied in vitro (124). Nuclear localization

of the receptor by Western blot has been reported, we investigated the posibility to

localize the receptor by immunohistochemical method within the tumor cells of NB and

PNET.

Materials and methods

Twenty-two cases of NB and four cases of PNET were investigated. In a

retrospective study basic clinical data, such as age, sex, location of the tumor,

chemotherapy and/or surgical removal irradiation and survival were registered (Table

1).

Among the children suffering from NB 14 were males (average age 3.40+0.80)

and 8 were female (average age 3.40+1.5 years). Three boys (2.5 and 10 years old) and

one girl (4 years old) had PNET. Seven neuroblastomas and two PNETS were in Evan’s

stage IV, four neuroblastomas were in Evan’s stage II, one neuroblastoma and one

PNET were in Evan’s stage I,and one PNET was in Evan’s stage II at diagnosis (Table

27

2). The tumor tissues were obtained by surgical excision before treatment from the

primary tumor, and after formalin fixation and paraffin embedding 8um thin sections

were cut.

Patient Sex Age

(Year) Chemotherapy Radio-therapy Location Operation Stage/Evans

1.NB F 0.8 Endoxan + ADR + steroid . OPEC None Mediastinum Part.res. St.IV.

2.NB F 1,2 NB-90 RG-C None Mediastinum Part.res. St.III.

3.NB F 8

Vac+OPEC+OJEC(RETINOID)+ Autotransplantation None

Retro-peritoneum Part.res. StIII.

4.NB F 1 None None R. adrenal gland

Total resection St.I.

5.NB F 6 OPEC/OJEC None Retro-peritoneum None St.III.

6.NB M 4 NB-90-RG-C 32 Gy Retro-peritoneum Inoperable St.IV.

7.NB M 1 OPEC+Newcastle+Autotranspl. None

Retro-peritoneum Inoperable St.III.

8.NB M 4 No clinical data/ foreign person/ None

Retro-peritoneum None St.IV.(?)

9.NB M 4 ALL-BFM-88 None Retro-peritoneum Part.res. St.IV.

10.NB M 6 OPEC 30 Gy R. adrenal gland Part.res. St.IV.

11.NB M 3 OPEC None Pelvic lymph node Part res. St.IV.

12.NB M 5 OPEC None Retro-peritoneum Inoperable St.IV.

13.NB F 6 VP-16+Carboplatin None Retro-peritoneum

Auto.bone.m transpl. St.IV

14.NB F 2 n.d. None Bone marrow Bone m. transplant St.IV.

15.NB M 8 OPEC/OJEC None Bone marrow Bone m. transplant St.IV.

16.NB M 3 n.d. None Retro peritoneum

Bone m. transplant NA

17.NB M 0.3 n.d. None Adrenal gland None NA

18.NB F 0.5 HR-NBL-1/ESIOP None Retro-peritoneum None St.IV

19.NB M 2 n.d. None Adrenal gland None St. I.

20.NB M 3 n.d. None Adrenal gland None St. I.

21.NB M 3 n.d. None Retro-peritoneum None St.IV.

22.NB M 1 n.d. None Mediastinum None NA

23.PNET F 4 OPEC/OJEC None Retro-peritoneum Part.res St.IV.

24.PNET M 2 NB-A-89 None Retro-peritoneum Part.res St.I.

25.PNET M 5 VAIA 90 Gy Right. forearm Part.res. St.IV.

26.PNET M 10 NHL-BFM-95 None Mediastinum Part.res. St.II

n.d. = no data;

Table 1.

28

All sections were examined with HE staining. Neuroblastomas were of the small

round cell type and PNET showed the characteristics described elsewhere (125).

ApopDetek (simply sensitive in situ Detection Kit, Dako, Denmark) was used to show

apoptosis (126).

Table 2.

Patient Diagnosis Sex Age (Year) Apoptosis index (%)

RAR-B index (%) Survival (months)

1. NB F 0.8 4,8 22 5 2. NB F 1,2 1 0 24 3. NB F 8 0 0 >24 4. NB F 1 0 0 11 5. NB F 6 0 0 16 6. NB M 4 2 19 13 7. NB M 1 0 10 20 8. NB M 4 0 9 >24 9. NB M 4 1 3 6 10. NB M 6 3,8 17 23 11. NB M 2,3 3,4 34 17 12. NB M 2,5 0 11 20 13. NB F 6 0 30 n.d. 14. NB F 2 0 10 n.d 15. NB M 8 0 90 n.d 16. NB M 3 0 0 n.d 17. NB M 0,3 0 80 nuclear

20 cytopl. n.d

18. NB F 0,5 0 85 cytopl. n.d 19. NB M 2 3 50 cytopl.memb. n.d 20. NB M 3 0 70 n.d 21. NB M 3 0 50 nuclear n.d 22. NB M 1 0 - n.d 23. PNET F 4 5 43 4 24. PNET M 2 19,5 68 25 25. PNET M 5 6,8 41 42 26. PNET M 10 4,1 16 >20

Sections were treated with proteinase K to make the tissue permeable to the

reagents of the labelling and detection procedure. The apoptosis index in the tumor

tissue was determined by examining 2000 tumor cells. The presence of RAR was

29

examined by immunoperoxidase reaction using RAR-B Ab (BIOMOL, Germany,

cat.No.SA-17). This antibody has been reported to show RAR in Western blot (127).

The sections were treated with proteinase K and, to inactivate endogenous peroxidase,

incubated in methanol and H2O2. Undiluted anti-retinoic acid receptor-B was applied as

primary antibody and was incubated overnight at –4 C. The secondary Ab-mouse

immunoglobulin was used in 1:100 dilution the next day- DAB (diaminobenzidine) was

used as a chromogen and methyl green as a counterstain.

The same procedure was performed with RAR-B diluted in 1:5 and 1:10 to test the

sensitivity of the reaction, since according to our knowledge this is the first attempt to

use immunohistochemistry to show RAR. RAR index in tumor tissue was determined

by examining 2000 tumor cells.

Results The clinical data of our

patients are shown in

table1. With the

exception of two cases

(case 3 and case 6), death

was due to progression of

the tumor 5-42 months

after the diagnosis,

irrespective of

localization, histological

types, or mode of therapy.

Only one case (case 3)

received RA therapy, this

patient is still alive.

Apoptosis was shown in

6 NB tumor samples

(table-2).

The apoptotic index was

zero or 1% in 8 children

Fig. 1. NB case scattered apoptotic cells. ApopDetec staining (x300).

Fig. 2. NB RAR positivity on cell surface. Anti-RAR-beta immuno- peroxidase (x300)

30

and relatively low (2-4%) in the other four cases, while it was higher (4.1-10.5%) in

PNET. The RAR index determined by immunoperoxidase reaction in NB was zero to

3% in five cases and 9-34% in seven children (table-2). Scattered apoptotic cells in a

section of neuroblastoma is shown in Figure 1.

RAR index in PNET was 16-68% in all four cases. As shown in table 2, apoptotic index

and RAR index varied parallel to each other, i.e., when apoptosis was frequent, the

RAR index was also elevated. The correlation coefficent between apoptosis and RAR

index was r = 0.47 according to Pearson-Bravis.

RAR positivity appeared in the nuclei of tumor cells both in NB and PNET when the

antibody was applied undiluted (figures 2 and 3), but if 1:5 or 1:10 dilution was also

applied, cell surface and slight cytoplasmic positivity could be detected in the same cell

(Figure 4, 5), along with nuclear positivity, probably due to better resolution of the cell

organelle. There was no evaluable relationship between apoptotic index, age, and stage.

Fig. 3. RAR positivity in nuclei of NB. Anti-RAR-beta immonoperoxidase (x150)

31

Discussion A variety of tumors have been studied for spontaneous and induced apoptosis.

Inducibility of apoptosis in tumor tissue by various drugs or irradiation is considered as

a possible predictive factor (121,122,128). The spontaneous apoptosis index in NB and

PNET has been found to be very low (129, 130). The possibility of inducing apoptosis

by retinoic acid (RA) treatment was first studied by Smith et al (124) in vitro. In their

experiment RA-induced apoptosis in cultured NB cells was studied and this effect was

linked to RAR. Since RA therapy has been administered in some cases of NB (131,132)

Fig. 4. PNET cytoplasmic RAR positivity. Anti-RAR-beta immunoperoxidase (x300)

Fig. 5. NB case Tumor cell nuclei are strogly RAR positive, anti-RAR-beta immunoperoxidase (x300).

32

detection of RAR may predict the effectiveness of RA treatment. Although nuclear

localization of the receptor by western blot (133) has been reported we demonstrated by

immunoperoxidase reaction that at least a certain proportion of the receptor may be

situated on cytoplasmic membranes.

As for NBs, not all were RAR positive in our study. The apoptotic index was low in

general compared to other types of tumors, but it was elevated in cases with a strong

RAR positivity. In all cases of PNET, however, a relatively high apoptotic index as well

as strong RAR positivity was found.

To the best of our knowledge, immunohistochemical demonstration of RAR has not

been reported. Our findings show that the RAR-B Ab can be used successfully also for

immunohistochemical purposes. The results suggest that spontaneous apoptotic activity

in NB may be caused by endogenous retinoic acid and mediated by RAR.

33

Tumors in Thyroid glands

Nodules in the thyroid have always commanded a great deal of attention because of

their fear of being cancerous. The female-to-male ratio is about 3 to 4:1. Most of these

solitary masses prove to be dominant nodules within a multinodular goiter, cysts, or

asymmetric enlargements of various non- neoplastic conditions, such as Hashimoto`s

thyroiditis. When such nodules prove to be neoplastic, in well over 90% of instances,

they are adenomas. Virtually all adenomas of the thyroid are present as solitary, discrete

masses. With rare exception, they all are derived from follicular epithelium and so

might all be called follicular adenomas (134). Microscopically, a variety of patterns can

be identified that recapitulate stages in the embryogenesis of the normal thyroid, and so

they have been divided into fetal, embryonal, simple, and colloid subtypes or, more

simply, into microfollicular and macrofollicular patterns. There is little virtue in these

classifications because mixed patterns are common, and ultimately all have the same

clinical and biologic significance. Numerous studies have made it clear that adenomas

are not forerunners of cancer except in the exceptional instance.

Malignant Tumors in Thyroid glands

Thyroid carcinoma is two to three times more common in middle aged women than in

men. Before puberty, there is no sex difference, and the female preponderance

disappears in postmenopausal life. Possibly relevant is the fact that most well-

differentiated thyroid carcinomas have estrogen receptors. The morphologic variants of

thyroid carcinoma with their approximate frequencies are as follows:

? Papillary carcinoma 75-85%

? Follicular carcinoma 10-20%

? Medullary thyroid carcinoma 5%

? Anaplastic carcinoma (rare)

Papillary Carcinoma, is the most common type of thyroid carcinoma, comprising 45%

to 70% of all cases (123,135). It is a slow-growing tumor that usually appears clinically

as a solitary thyroid nodule. The primary tumor is in most cases limited to the gland, but

34

especially in older people it may invade the surrounding structures, such as muscles, the

esophagus, or the larynx. The prognosis of papillary carcinoma is relatively good, with a

10-year survival rate in the order of 80% to 95% (135,136,137). Histologically papillary

carcinoma is composed of papillae, usually accompanied by follicules and sometimes

by solid sheets of cells. The papillae are often long and slender and have a fibrovascular

core that is usually thin but may be thick and hyalinized. The nuclei of papillary

carcinoma often overlap and have many typical features including a pale outlook, large

size compared with normal follicular cells, irregular outline with deep grooves and

cytoplasmic pseudoinclusions, rare mitosis, and an inconspicuous nucleolus

(138,139,140).

Follicular carcinoma comprises about one fourth of all thyroid carcinomas. It is more

common in females than in males and is a disease of middle and old age. Distant

metastases are common, occurring in as many as 25% of patients at diagnosis (141).

Their most common sites are the bones and lungs. Histologically the tumor is often

encapsulated. The capsule usually contains large, thin-walled blood vessels. Vascular

invasion -often to these vessels- is common, a finding that explains the frequent

occurrence of distant bloodborne metastases in follicular carcinoma. Follicular

carcinoma shows follicular differentiation but lacks the diagnostic features of papillary

carcinoma (142). Like follicular adenoma, follicular carcinoma is composed of

follicular, trabecular, or solid structure (143).The degree of follicular differentiation

varies, but usually the follicles are smaller and less well differentiated than the follicles

in papillary carcinoma. Follicular carcinoma is divided into two subtypes: minimally

invasive (also called encapsulated) and widely invasive (142). In the first subtype the

tumor is encapsulated, and only minimal vascular or capsular invasion is seen. In the

second subtype the tumor is either nonencapsulated or encapsulated with considerable

invasion. These two subtypes should be distinguished in prognosis (144) because the

10-year survival rate for the first type is 80% to 95% and for the second type 30% to

45% (135,136,145).

Medullary Carcinoma basically differs from other thyroid carcinomas in showing

evidence of C-cell differentiation. Like normal C-cells, the tumor cells produce and

secrete calcitonin. The tumor may also produce prostaglandins histaminase (146), and

express carcinoembryonic antigen (CEA) (147), and occasionally excretes 5-

35

hydroxytryptamine (5_HT) or adrenocorticotropic hormone (ACTH). Although most

medullary carcinomas occur sporadically, about 10% have a genetic background (148).

The sporadic carcinoma is generally unilateral but the familial tumor is usually bilateral

and multicentric and often accompanied by C-cell hyperplasia, which precedes the

development of medullary carcinoma (149,150). Also, the associated

pheochromocytoma is usually bilateral (151). Medullary carcinoma is rather rare,

comprising about 5% to 10% of all thyroid carcinomas. It is as common in men as in

women and is usually detected at middle age. The most frequent finding is a solitary

thyroid nodule, but sometimes enlarged cervical lymph nodes may be the first symptom.

Regional lymph node metastases are common; they have been detected in about half the

patients at the time of surgery (152). There are often irregular calcifications in the

stroma that may resemble psammoma bodies but do not show regular laminations. It is

not always possible to find amyloid in medullary carcinoma. In such cases the diagnosis

can be confirmed by use of silver stains for argyrophil cells (153) or

immunohistochemical methods based on the calcitonin content of the tumor cells (154).

Mixed medullary-follicular carcinoma (156,157) denotes a very rare tumor with

morphologic features of medullary carcinoma with immunoreactive calcitonin,

accompanied with morphologic features of follicular carcinoma with immunoreactive

thyroglobin.

Anaplastic carcinoma: Fortunately fewer than 5% of thyroid carcinomas fall into this

category. They tend to occur in elderly individuals, particularly in endemic goitrous

regions. There are basically three histologic paterns: 1) spindle cell carcinomas, 2) giant

cell leions, and 3) rarest of all the small cell carcinoma (155). All grow quite rapidly and

are usually large masses by the time they come to clinical attention.

II.Apotosis in Thyroid glands

Our studies on apoptosis in thyroid gland tumors

This study has been carried out by the co-operation of the:

National Institute of Oncology, Budapest, Hungary with the full support of Dr. Ilona

Peter, whom without her assistance the work would have been impossible. And the

36

Institute of Parthology, University of Debrecen, Debrecen, Hungary, where the help of

Prof. Dr. Szabolcs Gomba is deeply appreciated.

Introduction

Thyroid nodules are found in as many as 1% to 10% of the population, but malignant

tumors of the thyroid account for only about 1% of all cancer and 0.4% of cancer

related death (156). Females are affected three times as often as males (106). The aim of

this study was to measure spontaneous apoptosis and the expression of some oncogenes

which influence this process: Bcl-2 by securing cell survival (5), mutant P53 by

suppressing and bax by promoting cell death (22). With the knowledge that Bcl-2

inhibits apoptosis in a number of different cells and bax is antagonizing the protective

effect of Bcl-2 it becomes clear that prolonged survival of cells overexpressing Bcl-2 is

a factor in predisposition to malignancy (5). Nevertheless, investigation of these

oncogenes in benign tumors-adenomas could indicate a chance of malignancy in such

tumors.

Materials & Methods

Sixteen cases of thyroid carcinoma and nine cases of adenoma were investigated in a

retrospective study. Basic data (age, sex, tumor type) were registered (table 3.).

No Age

(year) Sex

Tumor type Apoptosis %

P53 %

Bax

%

Bcl-2 %

1. 24 F F. adenoma 0 2.6 12.7 Neg. 2. 35 F F. adenoma 0 4.9 1.9 Neg. 3. 44 F F. adenoma 0 0.9 0 Neg. 4. 39 F F. adenoma 0 0 11.9 Neg. 5. 45 F F. adenoma 0 33.1 43.5 Neg. 6. 55 F F. adenoma 0 5.1 0 59 7. 20 F F. adenoma 0 0.6 0 19 8. 50 F F. adenoma 0 0.3 9.2 47 9. 51 F F. adenoma 0 3.5 40.5 Neg. Subtotal 40 F. adenoma 0 5.7 13.3 41.7 Table 3.a.1. Percentage of P53, bax, bcl2 and apoptosis in thyroid adenoma

37

No Age

(year) Sex

Tumor type Apoptosis %

P53 %

Bax

%

Bcl-2 %

10. 64 F F.ad. carcinoma 0 94 12.3 Neg. 11. 50 F F.ad. carcinoma 0 22.8 4.8 2 12. 68 M F.ad. carcinoma 0 46.7 0 Neg. 13. 71 F F.ad. carcinoma 0 53.1 1.6 Neg. 14. 67 F F.ad. carcinoma 0 100 0 3 15. 55 M F.ad. carcinoma 0 81.7 1.1 Neg. 16. 17 M F.ad. carcinoma 0 100 4.2 Neg. Subtotal 56 F.ad. carcinoma 0 71.1 3.4 2.5

No Age

(year) Sex

Tumor type Apoptosis %

P53 %

Bax

%

Bcl-2 %

17. 64 F P.ad. carcinoma 0 0 3.4 Neg. 18. 44 F P.ad. carcinoma 0 100 5 Neg. 19. 43 M P.ad. carcinoma 0 0.6 4.6 Neg. 20. 17 M P.ad. carcinoma 0 0.6 0 Neg. 21. 11 F P.ad. carcinoma 0 26 0 2 22. 23 M P.ad. carcinoma 0 0 13.1 Neg. 23. 65 F P.ad. carcinoma 1% 53.1 3.2 Neg. 24. 42 M P.ad. carcinoma 0 96.1 0 Neg. 25. 72 F P.ad. carcinoma 0 0.5 1.2 4 Subtotal 42 P.ad. carcinoma 0 39.6 3.4 3

Average Age (year)

Tumor Type Apopto-sis %

P53 Box Bcl - 2

40 ? 12 F. adenoma 0 5.7 ? 10,4 13.3 ? 10,0 41.7 ? 20,5

56 ? 18 F.ad.carcinoma 0 71.1 ? 30,4 3.4 ? 4,6 2.5 ? 0,7

42 ? 22 P.ad.carcinoma 0 39.6 ? 44,3 3.4 ? 4,1 3.0 ? 1,4

The tumor tissues were obtained by surgical excision, and after formalin fixation

and paraffin embedding 8 ? m thin sections were cut. All sections were examined with

HE staining. The criteria described by Wyllie (3) were used to recognize apoptotic cells.

ApopDetec (simply sensitive In situ Detection System Kit, DAKO, Denmark) was also

used to show apoptosis. Sections were treated with proteinase K to make the tissue

Table 3.a.2. Percentage of P53, bax, bcl2 and apoptosis in thyroid follicular adenocarcinoma

Table 3.a.3. Percentage of P53, bax, bcl2 and apoptosis in thyroid papillari adenocarcinoma

Table 3.b. Average of P53, bax, bcl2 and apoptosis in thyroid tumors

38

permeable to the reagents of the labeling and detection procedure. The apoptosis index

in the tumor tissue was determined by examining 2000 tumor cells. The sections were

examined for mutant p53, bax and bcl-2 by immunoperoxidase reaction .

All antibodies to p53, bax and bcl-2 were the products of DAKO (Denmark) and were

used in dilution 1:100 overnight at 37 ? C. The sections were treated with proteinase K

and, to inactivate endogenous peroxidase, incubated in methanol and H2O2. The

secondary antimouse IgG (product of DAKO) was used in dilution 1:100 the next day.

DAB (diaminobenzidine) served as a chromogen and methyl green as counterstain. p53,

bcl-2 and bax positivity were determined by examining 2000 cells.

Results

In adenomas p53 positivity was 5.7% (fig. 6.), bax positivity was 13.3% and bcl-2

positivity was 41.7%. As for the follicular adenocarcinomas p53 showed 71.1%

positivity (fig. 7.), bax was positive in 3.4% and (Fig. 6.)

0

10

20

30

40

50

60

70

80

bcl2 bax p53

FollicularadenomaFollicularcarcinomaPapillarycarcinoma

Fig. 6. Percentage of bcl2, bax and P53 in thyroid tumors

?20,5

?0,7 ?1,4

?10

?4,6 ?4,1

?10,4

?30,4

?44,3

39

bcl-2 was positive in 2.5%. In

papillary adenocarcinomas

(Fig.7,8,9,) the value for p53

was 39.6%, for bax was 3.4%

positivity and for bcl-2 was

3%. Apoptosis was almost

absent in all cases.

Discussion

Data from this study on

mutant p53, bcl-2 and bax

expression in benign and

malignant thyroid tissue

reveal considerable results.

Mutant p53 is very low in

Fig. 7. Abundant P53 positivity in cell nuclei of a follicular adenocarcinoma. Immunperoxidase (x200)

Fig. 9. P53 positivity in papillary adenocarcinoma

Fig. 8. P53 positivity in follicular adenocarcinoma. Immunoperoxidase (x200)

40

adenomas compared to its

expression in papillary and follicular carcinomas. Bcl-2 expression is high in adenomas

and low in carcinomas according to our data. Bax expression on the other hand, is

relatively high in adenomas but still lower than bcl-2 and it is low in carcinomas.

In agreement with our results, bcl-2 and bax had been reported to be co-expressed in

aggressive thyroid carcinomas (159). As for the positivity of bax expression in papillary

carcinomas Branet et al (5) obtained similar results as described here by us. It has been

supposed that when bcl-2 is present in excess, cells are protected against apoptosis

(159,160,161). However, when bax is in excess, cells are susceptible to apotosis. As

apoptosis is concerned, only very few apoptotic tumor cells could be detected in our

study in any of the benign or malignant tumors. This may be relevant to the high

expression of bcl-2. On the other hand, high expression of bcl-2 in adenomas suggests

the susceptibility to transformation to malignancy. From a practical point of view,

determination of p53, bcl-2 and bax ratio in thyroid tumors may contribute to the

differentiation between adenomas and especially follicular carcinoma (7).

41

Fig. 11. P53 positivity in follicular adenocarcinoma. P53 immunperoxidase (x600)

Fig. 12. Bcl-2 positivity in follicular carcinoma. Bcl2 immunperoxidase (x300)

42

With the request of WHO-IARC (International Agency for Research on Cancer), after

the publication of our paper on thyroid tumours (Farid P, Gomba SZ, Peter I, Szende B.

Bcl-2, P53, and Bax in thyroid tumours and their relation to apoptosis. Neoplasm 2001;

48(4): 299-301 the following text was sent, along with a chart (fig.6) and a photo

(fig.11) and was accepted for publication in the WHO book on Pathology and genetics

of tumours of Endocrine organ.

Thyroid follicular carcinoma, Somatic genetic, expression

profiles/proteomics

Follicular carcinoma is a much more aggressive lesion than papillary carcinoma. This

cancer occurs more often in females with a peak incidence in the fifth and sixth decades.

Mortality rates are 70% at 5 years.

There is no sign of increased apoptosis, or programmed cell death, as defined by

morphological change in non-pathologic cell loss and relevant to a wide spectrum of

biology; in follicular carcinoma, however as shown by immunohistochemical method

(presumably mutant) p53, bax, and bcl-2 were expressed.

When bcl-2 is present in excess, cells are protected against apoptosis (4,5,7) since it

secures cell survival (2). Mutant p53 suppresses, while bax promotes cell death (6,1).

When Bax is in excess, cells are susceptible to apoptosis. BCL-2 and Bax had been

reported to be co-expressed in aggressive thyroid carcinoma (4).

From a practical point of view the determination of P53, BCL-2 and Bax ratio in thyroid

tumors, contributes to the differentiation between adenomas and especially follicular

carcinomas (3). In adenomas the rate of P53 positive cells was 5.7%, while it was

13.3% for Bax and 41.7% for Bcl-2 in the tumor cell. The identification of the above

mentioned oncogenes, and many more, is facilitated by proteomics, that studies the

proteins in general and in particular their changes resulting from various disorders or the

effect of external factors (8).

43

References

1. Basolo F, Pollina L, Fontanini G, Fiore L, Pacici F, Baladanzi A. Apoptosis

and proliferation in thyroid carcinoma: correlation with Bcl-2 and P53

protein expression. Brit J Cancer 1997; 74: 537-541.

2. Lumachi F, Basso S. Apoptosis, life through planned cellular death

regulating mechanisms, control systems, and relations with thyroid disease.

Thyroid 2002; 12 (1): 27-34.

3. Farid P, Gomba Sz, Peter I, Szende B. Bcl-2, P53 and Bax in thyroid tumors

and their relation to apoptosis. Neoplasm 2001; 48 (4): 229-301.

4. Manetto V, Lorenzini R, Cordon-Cardo C, Krajewski S, Rosai J, Reed CJ,

Eusebi V. Bcl-2 and Bax expression in thyroid tumors. Virch Arch 1997;

430: 125-130.

5. Muller-Hocker J. Immunoreactivity of p53, Ki67, and Bcl-2 in oncocytic

adenomas and carcinomas of the thyroid gland. Hum Pathol 1999; 30: 926-

933.

6. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with

a conserved homolog, Bax that accelerates programmed cell death. Cell

1993, 74: 609-619.

7. Pierotti MA. Bcl-2 protein expression in carcinoma originating from the

follicular epithelium of the thyroid gland. J Pathol 1994; 172: 337-42.

8. Fountoulakis M. Proteomics: Current technologies and applications in

neurological disorders and toxicology. Amino Acid 2001; 21(4): 363-81.

9. Czyz W, Kuzdak K, Pasieka Z, Timler D, Brzezinski J. P53, MDM2, Bcl-2

staining in follicular neoplasms of the thyroid gland. Folia Histochem

Cytobiol 2001.

44

III. Tumors of the Parathyroid glands Normal parathyroid glands: In adults, most of the parathyroid glands are composed of

chief cells, but these give rise to several transitional forms as will be evident

subsequently. The chief contain lipofuscin pigment and secretory granules of

parathyroid hormones (PTH). Sometimes these cells have a “water clear” appearance

owing to lake of glycogen. Oxyphil cell and transitional oxyphils are found throughout

the normal parathyroid either singly or in small clusters.

The metabolic function of PTH in supporting the serum calcium level can be

summarized as follows, as a short review for better understanding the dysfunctions and

hyperplasia of the parathyroid glands (158):

1. PTH mobilizes calcium from bone.

2. It increases renal tubular reabsorption of calcium, thereby conserving it.

3. It promote renal production of 1,25-(OH)2D3, active in intestinal

absorption of calcium.

4. It lowers the serum phosphate level by enhancing phosphaturia.

Adenoma

Almost always solitary adenomas average 0.5 to 5.0 gm in weight but maybe much

larger. Although two may be present concomitantly in the same or different glands,

nodular hyperplasia must be ruled out when more than one adenoma is present. As a

rule, normal parathyroid parenchyma contains evenly distributed cytoplasmic fat

droplets, whereas adenomas do not contain such a fat, and hyperplastic parenchyma has

reduced or absent fat (162). The adenomas are well-encapsulated, soft, tan to red

lesions. Most are composed predominantly of chief cells, but often they contain foci of

oxyphil and transitional cells usually present as islands within a background of the chief

cells. Although oxyphil cells are traditionally thought to be non-functional, rarely a

hyperfunctioning adenoma is composed of these cells. In larger lesions, there may be

areas of infarct necrosis or hemorrhage.

45

Primary hyperplasia

Primary hyperplasia may occur sporadically or in the MEN syndromes I and II a. The

hyperplasia may be either diffuse or nodular and most often is composed predominantly

of chief cells but sometimes water clear cells and, in almost all instances, islands or

nodules of oxyphils. Poorly developed, delicate, fibrous strands may envelope the

nodules. The cells in the diffuse hyperplasia or within the nodules are arranged in a

wide variety of patterns: solid sheets, nests, trabeculae, or sometimes follicular

structures. One of the more distinctive features of primary hyperplasia is the dispersal of

occasional fat cells throughout the hyperplastic areas, but overall the total amount of fat

is reduced (163,164).

Secondary Hyperplasia

The syndrome most often appears in patients with renal failure ( uremic

hyperparathyroidism) but also with marked vitamin D deficiency or osteomalacia. The

origin of hyperparathyroidism in renal failure is attributed to phosphate retention and

hypocalcemia leading to compensatory parathyroid hyperfunction. In addtion, the renal

disease may contribute to the hypocalcemia by 1) a reduction in the synthesis of 1,25-

(OH)2D3 with impaired intestinal absorption of calcium and 2) some poorly understood

state of skeletal resistance to the calcemic action of vitamin D and PTH. Uremic

hyperparathyroidism may be observed in the absence of hypocalcemia. Similarly,

vitamin D deficiency may directly stimulate PTH secretion. Whatever the precise

mechanism, it is clear that patients with renal failure and rarely the other conditions

mentioned develop secondary parathyroid hyperfunction with hyperplasia principally of

the chief cells. In many instances, the glands revert to normal if the basic clinical

derangement is brought under control by, for example renal transplantation. With long

standing secondary hyperplasia, however, reversion to normal may not occur,

contributing to the possibility that secondary hyperplasia may in time convert into

autonomous adenoma formation, sometimes referred to as tertiary hyperparathyroidism

(165,166).

46

Parathyroid carcinoma

Fewer than 5% of cases of PHPT are caused by carcinomas. These tumors present as

gray-white, irregular masses that sometimes exceed 10 gm in weight and enlarge

usually one parathyroid gland. Many are small, however, and readily mistaken for an

adenoma. Histologically, the neoplastic cell may be of variable size and shape but are

usually remarkably uniform and not too dissimilar from normal parathyroid cells. There

is general agreement that a diagnosis of carcinoma based on cytologic detail is

unreliable, and local invasion and metastasis constitute the only reliable criteria of

malignancy (167).

III. Apoptosis in parathyroid tumors Our studies have been done with the co-operation of the Department of Surgery and

Transplantation, Semmelweis University, Budapest-Hungary, our gratitude goes to Prof.

Dr. Ferenc Perner and dr. Gyula Végso.

Hyperplastic and adenomatous lesions of the parathyroid glands are not uncommon, but

malignant tumors of this gland are extremely rare (168). Hyperplasias may be primary

lesions but in most cases parathyroid hyperplasia is the consequence of chronic renal

failure. Adenomas may appear as a subject of both primary and secondary

hyperparathyroidism (HP), but the majority of these tumours appear without anamnestic

data on renal disease (169). Of course, renal damage developes regularly as an effect of

elevated serum Ca level in HP.

Differential diagnostic difficulties are well known concerning hyperplasia, adenoma and

carcinoma of the parathyroid gland. The most important sources refer to morphological

citeria and biological behaviour of the tumours (170). The significance of DNA diploidy

in indicating the benign or malignanat character of parathyroid tumours was studied by

Bocsi et al (171). They found that DNA index had no value in deciding the benign or

malignant character of a given sample.

A few but important data are available on p53, bcl-2 expression in parathyroid

hyperplasia, adenoma and carcinoma. bcl-2 and p53 expression was found both in

hyperplasias and adenomas but carcinomas failed to express bcl-2 (172,173). The role

of other gene products in the differential diagnosis of parathyroid neoplasia like P27,

cyclin D1, and Ki67 was also emphasized (174,175,176).

47

We investigated the mitotic and apoptotic activity and also the expression of P53, Bcl-2

and Bax in hyperplasia and benign as well as malignanat tumours of the parathyroid

glands. The aim of this study was to look for differences in this respect between the

above-mentioned proliferative alterations and further to find out whether the prevalence

of enhancing or inhibitory factors of apoptotic activity may explain the rarity of

parathyroid carcinomas.

Materials & Methods Clinical and laboratory parameters: The parathyroid lesions presented here were clinically diagnosed and surgically

removed at the Clinical Department of Transplantation and Surgery of the Semmelweis

University, Budapest Hungary. Altogether 107 patients were operated on because of

hyperparathyroidism (HP), 55 proved to be primary (PH), 52 suffered from secondary

hyperparathyroidism (SH). Clinical symptoms and laboratory parameters (serum Ca and

P, creatinine and parathormone level as well as radiological examination) leading to

diagnosis of HP were registered together with the period in time between the onset of

clinical symptoms or the start of hemodialysis or the date of renal grafting and the

diagnosis. Localisation and number of enlarged and removed parathyroid glands;

postoperative serum Ca and parathormone levels were also registered.

Histological studies:

All removed parathyroid glands were fixed in buffered neutral formalin and embedded

into paraffin. 8µm thin sections were cut and stained with hematoxylin and eosin.

Immunoperoxidase reactions to detect P53, Bcl-2 and Bax as well as TUNEL reaction

to show apoptosis were performed. Anti P53, anti-Bcl-2 and anti-Bax antibodies were

the product of DAKO (Glostrup, Denmark). The antibodies were diluted 1:100 and

applied overnight at 37ºC. Sections were treated with proteinase K, incubated in

methanol and H2O2. The secondary anti-mouse IgG (DAKO, Glostrup, Denmark) was

used in dilution 1:100 the next day. Diaminobenzidine (DAB) served as chromogen and

methyl green as counterstain. The TUNEL reaction was performed using Apop Detec

(Simply sensitive In Situ Detection System Kit, DAKO, Glostrup, Denmark) which

detects DNA fragmentation along nucleosomes and stain the nuclei of apoptotic cells.

48

Cellular constitution of the parathyroid lesions and mitotic index (cosidering 1000-cells)

were determined using H&E stained sections. The criteria described by Roth (168) were

applied to differentiate between hyperplasia, adenoma and carcinoma. Apoptotic index

was established after TUNEL reaction considering 1000 cells. Both mitotic and

apoptotic indices were expressed in percent. bcl-2 and bax, as well as P53 expression

was studied considering chief cells, oxyphil cells, and clear cells. Positivity was stated if

50 percent or more of a cell type showed cytoplasmic or nuclear reaction with DAB.

Results

The age and sex distribution and the per cent of adenomas, hyperplasias and carcinomas

are shown in fig. 13.a and 13.b. The overwhelming majority of patients with primary

lesions was female. SH occurred in females and males in nearly equal number. The age

of patiens suffering from PH was in average higher than that of patients with secondary

hyperparathyroidism.

0

10

20

30

40

50

60

70

80

a hy cc

adenoma

hyperplasia

carcinoma

0102030405060708090

a hy m

adenoma

hyperplasia

mixed (adenoma andhyperplasia)

Total: 55 6 males 49 females Age: 23-70 32-76 (59,2) (60)

Total: 52 25 males 27 females Age: 14-71 20-71 (49,5) (43,3)

Fig. 13.a. Primary hyperparathyroidism age, sex distribution and percent of adenomas, hyperplasias and carcinomas

Fig. 13.b. Secondary hyperparathyroidism age, sex distribution and percent of adenomas, hyperplasias and mixed lesions

49

The majority of primary lesions were adenomas and only three carcinomas were found.

In case of secondary lesions hyperplasia was the most common finding.

The clinical symptoms and events leading to the diagnosis of HP are shown in table 4.a.

PH resulted in nephrolithiasis and in osteoporosis as well as other bone and joint

alterations in a high number of cases. SH was predictable by the fact of pre-existing

chronic renal failure treated by dialysis or renal transplantation. We have to mention

that no MEN cases and no vitamin D deficiency or marked osteomalcia cases were

found in our material. The most characteristic laboratory findings are listed in table 4.b.

PH SH

Symptoms and

events

No of cases Symptoms and events No of cases

Nephrolithiasis

Osteoporosis

Bone fracture

Bone-joint pain

Elevated se Ca, P

gastrointestinal

23

25

3

13

9

5

Osteoporosis

Bone-joint pain

Bone fracture

Nephrolithiasis

Soft tissue calcification

Gastrointestinal

Renal graft

Dialysis

Renal graft plus renal

failure

Renal failure

14

16

5

1

3

2

26

19

6

1

Table 4.a. Clinical symptoms and events leading to the diagnosis of HP

50

PH SH

Laboratory

findings

Pre-operative Post-

operative

Pre-operative Post-operative

Se Ca (mmol/l)

Se P (mmol/l)

Se PTH (pg/ml)

Se creatinine

(dialysis)

(? mol/l)

Se creatinine

(renal graft)

(? mol/l)

3(2,73-3,5)

0,83 (0,59-1,24)

247,9 (68,6-1301)

2,23 (1,4-2,62)

20,3 (1,86-49,2)

2,74 (2,2-3,21)

1,71 (0,63-3,0)

821,9(77,3-2510)

769 (180-1194)

183 (95-338)

2,14 (1,44-2,7)

50,6 (1,2-251)

High serum Ca and P levels as well as PTH level were registered in both PH and SH

cases, higher values being charateristic to SH. Preoperative serum Ca and PTH levels

reflected high PTH activity. Definitive preoperative diagnosis was secured by

ultrasound and/or CT imaging as well as by isotope scanning (Table 5.).

PH SH

Ultrasound 35 34

CT 39 41

Isotope 54 48

One of the above 7 4

Two of the above 24 24

Three of the above 24 24

Table 4.b..Characteristic laboratory findings in HP patients

Table 5. Number of cases diagnosed and localized by ultrasound, CT and isotope techniques

51

In 34 cases of PH postoperative Ca administration became necessary. Subcutaneous

autotransplation of parathyroid tissue was performed postoperatively in 13 cases of SH.

Serum creatinine level was high in nearly all SH patients with chronic renal disease.

The time gap between the onset of symptoms in PH was relatively long (5.6 years)

calling the attention to the difficulties of diagnosis of HP. The period between the start

of diagnosis on the renal grafting was in average 4,2 (Table 6.) and 9 years,

respectively.

The number of the removed parathyroids is indicated in table 7. Primary lesions were

solitary in the majority of cases and were localized in the inferior right and left

parathyroids.

PH 5-6 (0,5-30)

SH (start of dialysis) 4-2 (0,25-13)

SH (renal grafting) 9 (1,5-28)

PH SH

No of enlarged

parathyroids

No of cases No of cases

1

2

3

4

48

2

4

1

5

8

16

23

Postoperative complications such as bleeding, infection,recurrent laryngeal nerve

damage occurred in altogether 6 cases. Regarding HP all operations for PH and all but

two operations for SH proved to be successful.

Table 6.Time gap between the onset of clinical signs and events and the diagnosis of HP (years)

Table 7.Number of the parathyroid lesions

52

Histological examination of the removed parathyroids showed the structural and cellular

features of hyperplasia, adenoma or carcinoma (see Fig 13 a, b).

The typical histological appearance of an adenoma and the immunostaining for

parathormone are shown in figure 14. a. and 14. b.

Fig. 14. a. Parathyroid adenoma HE (x300)

Fig. 14. b. Parathormone adenoma. Parathormone immunperoxidase (x300)

53

The cellular constitution of adenomas and hyperplasia is shown in fig. 15. Chief and

oxyphil cells were both present in the lesions and clear cell component was also found

in hyperplasia. No difference could be detected between the primary and secondary

lesions in this respect.

Apoptotic index was equally low in adenomas and hyperplasias and so was the mitotic

ratio. The carcinomas showed slight elevation in mitotic and apoptotic activity (fig. 16).

0102030405060708090

adenoma hyperplasia

oxyphilchiefclear

0

1

2

3

4

hyperplasia adenoma carcinoma

apomito

±0,1

±0,02

±0,15

±0,02

±0,2

±0,3

Fig. 16. Apoptotic and mitotic index.

Fig. 15. Distribution of cells types in hyperplasia and adenoma.

±7 ±6

±4

±7

±3

54

The ratio of P53, Bcl-2 and Bax expression was studied considering chief cells, oxyphil

cells and clear cells. Hyperplasias showed p53 positivity in the oxyphil cells in 10% of

the cases, chief cells were positive in 5 percent. The positivity was nuclear in general,

but in some cases cytoplasmic positivity was seen. The ratio of bcl-2 positivity was 60

percent in the oxyphil, 20 percent in the chief and 10 percent in the clear cells, on

average.

The bax expression was ten percent higher in all types of cells, compared to bcl-2

expression (fig. 17.a.,17.b.).

Fig. 17.a.

Fig. 17.b.

55

Figure 18.a. shows Bcl-2 positive cells in hyperplasia. bax positive cells in

hyperplasia are shown in figure 18. b. Co-expression of bcl2 and bax, as well as p53

was registered in most of the positive cases. Adenomas were positive for p53 in 15

percent of the cases, considering oxyphil cells and in 5 percent of the cases

considering chief cells. Positivity appeared in the nucleus in the majority of cases

but in few cases cytoplasmic positivity was observed (Fig. 19.a, 19. b).

Fig. 18.a.parathyroid hyperplasia, Bcl2, immunoperoxidase (x300)

Fig. 18.b. Bax positivity in hyperplasia. Bax immunperoxidase (x300)

Fig. 19.a. P53 nuclear positivity in parathydoid adenoma. P53 immunperoxidase (x300)

56

The expression of bcl-2 and bax in the oxyphil cells of adenomas was equally high and

a relatively high expression of bcl-2 and bax was present in the chief cells. Figure 20.a.

and 20. b. shows bcl-2 and bax positive cells of adenomas, respectively. Co-expression

of the three genes was characteristic to adenomas as well.

Fig. 20.a. Bcl2 positivity in parathyroid adenoma. Bcl2 immunperoxidase (x300)

Fig. 20.b. Bax positivity in parathyroid adenoma. Bax immunperoxidase (x600)

57

The few cases of carcinomas showed nuclear p53 positivity (Fig. 21.a.) and the

cytoplasm was negative for bcl-2 and bax (Fig. 21.b.).

Fig. 21.a. P53 nuclear positivity in parathyroid cancer. P53 immunperoxidase (x 300)

Fig. 21. b. Bcl2 negativity in parathyroid cancer. Bcl2 immunperoxydase (x600)

58

Discussion The results of our study on a relatively large number of parathyroid hyperplasias and

adenomas clearly indicate that mitotic or apoptotic activity can not differentiate between

these two pathological entities. Both mitotic and apoptotic indices were equally low in

hyperplasias and adenomas. In accordance with the study of Ricci et al (172) p53 and

bcl-2 expression was found in hyperplasias as well as adenomas. The percent of cases

considered as positive for p53 or bcl-2 was slightly higher in adenomas, but differential

diagnosis can not be based on p53 or bcl-2 immunostaining. The same may be applied

to bax expression. According to our knowledge expresion of bax has not been studied

yet in proliferative parathyroid lesions. The most interesting finding in our study was

the co-expression of bcl-2 and bax in most hyperplasias and adenomas. Oxyphil cells

and chief cells were studied separately regarding p53, bcl-2 and bax expression.

Oxyphil cells showed positivity for these gene products in a significantly higher

proportion of cases than chief cells or modified (clear) chief cells. This fact may be due

to the abundance of mitochondria in oxyphil cells. The appearance of p53 positivity in

the cytoplasm of both hyperplastic and adenomatous lesions may point to the increased

activity of inhibitor of P53 amino acid terminal nuclear signal (177) which means an

increased defense against transport of mutant P53 protein into the nucleous. The

antibody used by us shows predominantly mutant P53 by immunostaining. Nuclear

positivity of some of the adenomas may be a sign of the tendency towards malignant

transformation.

The very low number of carcinomas does not allow us to draw definite conc lusions

comparing these tumours with adenomas. In accordance with the litrature (178,179)

mitotic as well as apoptotic activity were slightly elevated in carcinomas. Nuclear p53

positivity and the abscence of bcl-2 as well as bax expression in carcinomas are in

accordance with the findings of Stojadinovic et al (173). Co-expression of p53, bcl-2

and Bax may be a factor responsible for the low mitotic and apoptotic activities well as

for the very low rate of malignomas in the parathyroid glands.

Our clinical findings may contribute to the differential diagnosis of hyperplasia and

adenoma of the parathyroid glands. Single lesions are more likely adenomas than

multiple ones. Adenomas occurred overwhelmingly in females. The gender distribution

in case of hyperplasia was practically even. The average age of patients with PH was

close to 60, SH occurred in earlier years of life.

59

Serum Ca and PTH levels were significantly higher in hyperplasias compared to

adenomas. Chronic renal failure, dialysis or renal grafting are anamnestic data pointing

to SH and the likelihood of hyperplasia. The presence of bone and joint lesions in both

PH and SH and the very long time between the onset of these symptoms and the

surgical removal of the parathyroid lesions calls attention to the importance of

considering the possibility of HP first of all in case of osteoporosis.

60

Conclusions

In Neuroblastomas and PNET apoptotic index and RAR index varied parallel to

each other, i.e., when apoptosis was frequent, the RAR index was also elevated. As for

NBs, not all were RAR positive in our study. The apoptotic index was low in general

compared to other types of tumors but it was elevated in cases with a strong RAR

positivity. In all cases of PNET, however, a relatively high apoptotic index as well as

strong RAR positivity was found.

To the best of our knowledge, immunohistochemical demonstration of RAR has not

been reported yet. Our findings show that the RAR-ß Ab can be used successfully also

for immunohistochemical purposes. The results suggest that spontaneous apoptotic

activity in NB may be caused by endogenous retinoic acid and mediated by RAR.

From the practical point of view the treatment of patients with RA was dependent on the

result of our examination and it the case of positivity for the RAR the treatment was

carried out nt the clinic.

Having examined p53, bcl-2 and bax in different tumors of the Thyroid, a new

dimension has been observed. Mutant p53 is very low in adenomas compared to its

expression in papillary and follicular carcinomas, and mutant p53 suppresses cell

death. Bcl-2 expression is high in adenomas and low in carcinomas according to our

data, and bcl-2 secures cell survival. bax expression on the other hand, is relatively high

in adenomas (but still lower than bcl-2) and low in carcinomas, and bax promotes cell

death.

The high expression of Bcl-2 explains the low ratio of apoptotic cells in the examined

thyroid tumors, benign and malignant. The high expression of Bcl-2 in adenomas

suggests the susceptibility to tranformation to malignancy. Although few apoptotic

tumor cells could be detected in our study in any of the benign or malignant tumors this

may be relevant to the high expression of bcl-2, since bcl-2 is known to secure cell

survival.

From the practical point of view, determination of P53, Bax and Bcl-2 ratio in thyroid

tumors contributes to the differentiation between adenomas and especially follicular

carcinomas.

As to the parathyroid lesions the overwhelming majority of patients with primary

lesions were female. Secondary hyperparathyroidism occured in females and males in

61

nearly equal numbers. The age of patients suffering from PH was on average higher

than that of patient s with SH. The majority of primary lesions were adenomas and only

three carcinomas. In case of SH, hyperplasia was the most common finding.

Apoptotic index was equally low in adenomas and hyperplasia and so was the mitotic

ratio. The carcinomas showed slight elevation in mitotic and apoptotic activity.

The results of our study on a relatively large number of parathyroid hyperplasias and

adenomas clearly indicate that mitotic or apoptotic activity cannot differentiate between

these two pathological entities. Bcl-2 and bax and in some cases p53 expression was

found in hyperplasias as well as adenomas. The percent of cases considered as positive

for p53 or bcl-2 was slightly higher in adenomas, but differential diagnosis can not be

based on p53 or bcl-2 immunostaining.

The same may be applied to bax expression (according to our knowledge expresion of

bax has not been studied yet in proliferative parathyroid lesions).

The most interesting finding in our study was the co-expression of bcl-2 and bax in most

of hyperplasias and adenomas.

Spontaneous apoptosis is generally low in endocrine cells, but overall knowledge in this

field reveals that hormones produced by different endocrine cells like PTH or LH

induce apoptosis. This by itself probably can build up a protective mechanism in

endocrine cells against apoptosis. The great emphasis in our study had been given to

shed light over this subject and has called our attention to the co-expression of bax and

bcl-2 and its relation to the protective mechanism against apoptosis in endocrine tumors.

62

Abbreviations

NB, Neuroblastoma

PNET, Primary neuroectodermal tumors

PCD, Programmed Cell Death

INPC, International Neuroblastoma Pathology Committee

NTs, Neuroblastic Tumors

OPEC, Oncovin+Cisplatin+Etoposid+Cyclophosphamid

OJEC, Oncovin+Cyclophosphamid+Etoposid+Carboplatin

VAIA, Vincristin+Actinomycin/Adriamycin+Ifosfamid

VAC, Vincristin+Actinomycin/Adriamycin+Cyclophosphamid

ALL-BFM-88/NHL-BFM-95,

Prednisolon+Vincristin+Daunorubicin+Cyclophosphamid+L-

Asparaginase+Leupurin+Metotrexat.

NB-A-89, Dacarbazin+Adriamycin+Mustarnitrogen+Vincristin

Newcastle, Vepesid+Ifosfamid/Vincristin+Cisplatin/Vincristin+Ifosfamid+Adriamycin

RAR, Retinoic Acid Receptor

DAB, Diaminobenzidine

PTH, Parathormone

PH, Primary Hyperparathyroidism

SH, Secondary Hyperparathyroidism

63

Acknowledgement

It seems that the journey has ended, but as Baha’u’llah said in one of his masterpiece

the seventh valley is the valley of true poverty and absolute nothingness, he added that

these journeys have no visible ending in the world of time, but the severed wayfarer- if

invisible confirmation descend upon him and the Gurdian of the Cause assist him-may

cross these seven stages in seven step, nay rather in seven breaths.

The invisible ending is when one finding leads us to search for another, and that is the

beauty of research work. The invisible confirmation was deeply felt, and the heart-

warming fact that I indeed was not alone on this journey was a daily experience. I

would like to express all my respect, gratitute and appreciation to beloved Professor

Bela Szende without whom none of the my imagination would take the color of reality.

His patience and his love to the research work has been exemplary. I greatly appreciate

the possibilities given by Professor László Kopper the head of the department for

fullfiling my dream in the 1st Department of Pathology and Experimental Cancer

Research. My utmost thank to Professor András Jeney for his continous support. I

would like to thank my dear husband Dr.Ajang Farid, for being extremely supportive

and for loving me for who I am. My hearfelt appreciation goes to our assistant in the

laboratory Dr.Paczolay Gyozone and dear Mrs.Erzsebet Kiss who has been a great help

with the computer work. I also would like to thank Mr.Andrew Singer for proof reading

my paper with the accuracy he did.

64

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