Genetic predisposition to cancer with special reference to mutagen sensitivity

IN VITRO CELLULAR ~ DEVELOPMENTAL BIOLOGY Volume 23, Number 9, September 1987 �9 1987 Tissue Culture Association, Inc.

I N V I T E D R E V I E W

G E N E T I C P R E D I S P O S I T I O N T O C A N C E R W I T H S P E C I A L R E F E R E N C E T O M U T A G E N S E N S I T I V I T Y

T. C. HSU'

Section of Cellular Genetics, The University of Texas, M. D. dnderson Hospital and Tumor Institute at Houston, Houston, Texas 77030

iReceived 15 June 1987; accepted 23 June 1987t

I NTRODUCT1ON

During the past decade, considerable progress has been made in the field of cancer genetics and cytogenetics. One of the reasons for the advancement has been technical improvement in identifying human and mammalian chromosomes and subdivisions of chromosomes. A reciprocal translocation of segments similar in length cannot be recognized by examining unbanded chromosomes; but with banding , especially high- resolution banding Il l , identification of such a rearrange- ment becomes not only feasible but also accurate in terms of breakpoints. The techniques have been effectively employed to compare neoplasms of the same pathology and have positively aided the conclusion that at least in some types of cancer the same chromosome aberration or aberrations prevail.

Cooperative work among geneticists, cytogeneticists, biochemists, molecular biologists, and clinicians has shown that special genetic changes may be etiologically responsible for neoplasia in target cells. Evidence also demonstrates that at least in some cancers specific genetic lesion(s) can be inherited. In another area of investigation, we learned that persons with some syndromes exhibit a high rate of spontaneous chromosome instability and are cancer-prone. The correlation between genetic instability and cancer led to another phase of investigation, viz, mutagen sensitivity and cancer.

In this article, I shall try to present the concept of cancer genetics and to discuss mutagen sensitivity in more detail; but first we must briefly cover the more basic topics because of the interrelationships of these subjects. In view of the current explosive research activities in human cancers and the availability of convincing examples, this discussion will be confined to human neoplasms. The concepts and conclusions may also be applicable to tumorigenesis in other mammals.

Genetic and Cytogenetic Specificity in Neoplasms

The subject of genetic and cytogenetic specificity in neoplasms is well known among cancer geneticists. Through studies on hematologic malignancies, specific

1 To whom requests for reprints should be addressed at Box lt~l, M.D. Anderson Hospital, Houston, TX 77030.

translocations such as tt8;14~ in Burkitt 's lymphoma (2} and t{9;221 in chronic myelogenous leukemia tCML~ (3} have been discovered and confirmed in numerous cases. The translocations are not only specific for the chromosomes involved but are also specific for the break sites.

Several important inferences can be drawn from these findings, a~ Conversion of a normal cell into a neoplastic cell requires a specific genetic alteration or alterations. However, a specific genetic alteration necessary for the malignant property of one cell type is conceivably harmless in another cell type. bl The specificity of genetic alteration that triggers neoplastic changes may depend on the stage of development of the target cells, c~ The specific mutational events in Burkitt 's lymphoma and CML occur in persons with normal chromosomal constitution. Thus, the abnormalities are acquired and not inherited. This suggests that chromosomal mutations occur frequently in somatic cells during a person's life, but that only the right combination occurring in the right cell would convert a normal cell to a malignant one.

Technical difficulties have hindered the search for specific cytogenetic alterations in various solid tumors. Nevertheless, some examples such as the monosomy or partial monosomy of a G-group chromosome have been found in meningiomas before the development of chromosome banding techniques (4t. Subsequent banding analysis showed that there was no translocation. The missing chromosome was No. 22. The situation is analogous to the now well-known interstitial deletion of 13q in retinoblastoma. In the first case of retinoblastoma described by Lele et al. (5L one D-group chromosome was found to be distinctly shorter than the rest, but the authors considered the abnormality and the neoplasm a coincidence. Additional investigations by other workers (6,7~ not only identified the anomaly as an interstitial deletion of chromosome 13 but also established this aberration as the etiologic factor of retinoblastoma. Similar specific anomalies have been identified in aniridia-Wilms' tumor. A deletion involving the l lp13 band seems to be the common denominator ~8-10t.

Recent research activities in cancer cytogenetics have made significant progress in quest of specific genetic lesions of solid tumors, including renal cell carcinoma [11,12L malignant melanoma (13,14L breast cancer I15L and others. Most of the tentative conclusions were derived

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from observations of long-term cell lines in which numerous cytogenetic changes had occurred. To deter.. mine the chromosome changes that may represent the initiation of a particular type of neoplasm, one must compare the chromosome constitutions of a large number of cell lines and see if a particular aberration or aberrations are shared by most, if not all, of the cell lines. This type of investigation is tedious, and the interpreta- tions are empirical. However, before better methods are developed, this seems to be the most logical approach. Two teams of investigators (13,14) have independently concluded that the terminal segment of 6q may contain a key gene for the development of malignant melanoma. The terminal segment of 6q or the entire 6q was found to be missing or replaced with another chromosome segment via translocations.

Probably the studies in breast tumors have been the most fascinating and most controversial. In analyzing a number of cell lines derived from human breast cancer, Cruciger et al. (15) found, among numerous chromosome aberrations, that all shared a break in the lq21 region. Perhaps this aberration represents the initial change toward neoplasia of the breast tissue; but in the breast tumor cells, subsequent investigations revealed another set of chromosome aberrations. In a pleural effusion sample from a woman with a breast tumor, direct fixation yielded sufficient metaphases for critical cytogenetic analyses (16). The cells had a stemline number of 35. In addition to the low stemline number, G-band preparations revealed that the cells also contained all six marker chromosomes characteristic of the HeLa cells. The fact that the cells were never cultivated and that the low stemline was never recorded in HeLa indicated no cell line contamination. This finding raised an interesting question: Could HeLa markers be found in other cases of breast cancer? Satya-Prakash et al. (17) studied eight breast cancer lines and reported that six of them exhibited at least one HeLa marker chromosome. An expanded study (18), using not only long-term cell lines but also short-term cultures and direct biopsy specimens, showed HeLa markers in practically every specimen. I t seems that HeLa marker chromosomes may be characteristic for breast cancer cells. Thus, it becomes a provocative notion to challenge the classic belief that the HeLa cell line was derived from a cervical carcinoma. As additional negative evidence, cells from three human cervical carcinoma lines failed to show any HeLa markers (19,20).

Admittedly, the data on solid tumors are at present insufficient and inconclusive; but we expect progress to be made as techniques for short-term culture and direct biopsy handling are improved. In the not too distant future, geneticists may find that each neoplasm starts with a specific genetic alteration and that many such lesions can be detected by cytogenetic methods.

Congenital Chromosomal Aberrations and Predisposition to Cancer

The t(8;14) translocation of Burkitt 's lymphoma and the t(9:22) translocation of CML are acquired at some stage of

postnatal li/e. Apparently, in these neoplasms a single translocation at two specific gene loci disturbs the structure and function of vital genes such that the genetic alteration behaves as a dominant mutation, because the genes in the homologous chromosomes apparently remain un- changed. However, many genetic changes that cause malignancy are of the recessive type, i.e. both genes in the homologous chromosomes must maffunction or be absent. In such cases, a congenital mutation or deletion in one of the homologous chromosomes does not induce a neoplastic conversion until the homologous allele also changes. Thus, a person carrying a constitutional defect characteristic of a particular tumor is not born with a tumor.

Briefly, the two-step mutation hypothesis of Knudson (21) can be paraphrased as follows: A target gene must be mutated in both homologous chromosomes to change a normal cell into a malignant cell. The probability of having mutational events occurring in both genes simultaneously is obviously negligible. Thus, two genetic changes, in two steps, are the minimum requirements. However, if one mutation is inherited as a constitutional defect, then only one mutational event is needed to accomplish the malignant conversion. Knudson based his theory on statistical analyses of familial and clinical information on retinoblastoma. He found that the hereditary type of this neoplasm had early onset and often with multiple loci, whereas the nonhereditary type showed late onset and a single lesion. In other words, in hereditary retinoblastoma, one mutation was in existence in the prezygotic stage; therefore, only one additional mutation is required to convert a normal cell into a malignant one, resulting in early onset and multiple lesions. In the nonhereditary type, both homologous genes must mutate; hence the delay of onset.

The discovery of a congenital interstitial 13q deletion in several children with hereditary retinoblastoma gave Knudson's hypothesis the best support on a physical basis. Here a gene is missing by a chromosome deletion, so that the first step is the prezygotic absence of one of the two target genes, leaving only one gene in the homologous chromosome responsible for the development and maintenance of the retinoblasts. When that gene is mutated, cancer may be initiated. In di/ferent cases, the 13q deletions were not identical in length and in breakpoints; but all of them contained the 13q14 band, suggesting that the gene in question is located in the vicinity of that band.

Children can inherit the 13q deletion from parents with balanced translocations (22,23). In one family, the proband showed a 13q deletion, but both parents were normal (23). Cytogenetic analysis revealed that the mother had a 13q deletion, but the deleted segment was inserted into one of the chromosomes N2. Meiotic segregation gave the gamete 13q- and a normal chromosome N2. The proband was, therefore, predisposed to develop retinoblastoma.

The fact that most retinoblastoma cases studied did not exhibit the 13q deletion does not argue against the two-step mutation hypothesis, because a gene mutation accomplishes the same effect as a gene loss. The positive

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13q deletion cases are strong testimonies to the relationship between the genetic defect and the tumor. One may argue that the retinoblastoma cases are unique. Whether a generalized principle can be established requires confirmation from other neoplasms. Therefore, the aniridia-Wilms' tumor combination became extremely important. Knudson and Strong {24~ found that the pattern of inheritance of Wilms' tumor almost paralleled that of retinoblastoma. In hereditary Wilms' tumor, a second phenotype, aniridia, seemed to be always associated with genetic susceptibility to Wilms' tumor. Therefore, the discovery of the l l p interstitial deletion in aniridia-Wilms' tumor 125) gave the theory of genetic etiology of cancer the strongest support. When the information on retinoblastoma and Wilms' tumor is combined, one can confidently conclude that cancer is indeed initiated by genetic changes of target cells and that the genes in question are specific for specific tissues.

The discovery of constitutional cytogenetic aberrations in normal cells of patients with retinoblastoma and Wilms' tumor suggest that chromosome aberrations etiologically related to other neoplasms, especially those with a hereditary mode, may also be found in normal cells of the patients and their relatives. The chromosome aberrations are not expected to be limited to deletions and are not expected to be found in a large number of cases. However, even a low percentage of cases would be most suggestive if the aberrations have some degree of consistency. I t is, in a way, working backward to find possible etiologic lesions of cancer cells by looking at the normal cells, because in normal ce l l s constitutional abnormalities, if present, would not be as complex as the aberrations contained in the tumor cells. This approach has begun to provide useful information in studies of several types of neoplasms.

Renal cell carcinoma. Cytogenetic analyses of family members with hereditary renal cell carcinoma (261 showed a constitutional translocation between chromosomes N3 and N8 in lymphocytes of all available patients as well as three nonsymptomatic female members. The correlation between the chromosome aberration and the disease seemed strong. But if this translocation represented the first-step mutation for the initiation of renal cell carcinoma, then the three nonsymptomatic members belonged to the at-risk group. Indeed, these individuals developed kidney lesions shortly afterward. Thus, the t(3;8) should be regarded as the genetic factor predisposed to renal cell carcinoma, at least in that family. Several questions arise from this particular study: a) Is this genetic anomaly characteristic of all cases of renal cell carcinoma or is it only limited to this family? b) If it is a crucial genetic change that causes this renal cancer, must the translocation be specifically limited to chromosomes 3 and 8, as in t(8;14~ of Burkitt 's lymphoma; or is only one chromosome vital, but the recipient chromosome can vary? c) Must the breakpoints also be specific, as in Burkitt 's lymphoma cases?

Cytogeneticists began to examine the chromosomes of renal cell carcinomas in both the tumor ceils and the normal cells of the same patients. Available data showed

that in the tumor cells, the 3p13-14 region was always involved in rearrangements, but the partner chromosome could be N8, N i l , N6, N16, or N17 t11,27,28L However, the iymphocytes of most of these patients seemed normal. These findings indicate that breakage in the 3p13-14 region may be inherited or may occur de novo in the target tissue, but it seems to be critically associated with the disease. Inasmuch as renal cell carcinoma has familial and sporadic types, it is conceivable that congenital aberrations may be present only in the hereditary variety. In the nonhereditary variety, the 3p aberration occurs de novo in the target tissue. Therefore, lack of aberrations in normal cells is not evidence against the cytogenetic specificity of this neoplasm.

Breast cancer. Practically all cell lines derived from breast cancer exhibit aberrations with a breakage at lq21. However, no record of the normal cells of these patients is available. Breast cancer is well known to have hereditary and nonhereditary types. Thus, one would not expect to find a large number of constitutional chromosome abnormalities in cases of sporadic breast cancer. If constitutional chromosomal lesions do occur, they are more likely to be found in normal cells of family members with familial breast cancers. In several families, S. Pathak and his associates (unpublished data~ discovered a paracentric inversion at the lq21 locus. The other breakpoint of the inversion is at the C-band zone. The inversion divides the lq C-band into two zones with a euchromatic band between them. Linkage studies by Anderson et al. (281 on hereditary breast cancer also implicated chromosome lq.

T.cell leukemia. A number of cases of T-cell leukemia have been found by cytogeneticists to have a constitutional Robertsonian fusion between chromosomes N13 and N14, but other persons with the same translocation have never developed that neoplasm. Most investigators have regarded this correlation as coincidental. The picture became somewhat clearer when Pathak (29~ discovered a family in which a constitutional t(14;9) was found in three members: the mother, the daughter, and the son. The son had T-cell lymphoma. High-resolution banding identified the breakpoint on chromosome N14 at band 14q12. In retrospect, Pathak reexamined the chromosomes of a family with constitutional Rb~13;14~ and subsequent T-cell malignancy in one member. High-resolution banding showed that the breakpoint at chromosome N14 was also at band 14q12. Pathak hypothesized that in Rb(13;14) several breakage and restitution patterns are possible, but superficially the morphology of the fused metacentric chromosome is identical unless high-resolution banding analysis is done, Only when the breakpoint at 14q12, where the T-cell receptor gene is located ~30L may a person be predisposed to developing T-cell malignancy.

The brief accounts presented above suggest the following tentative conclusions: aj Each neoplasm may be initiated by a change at a specific gene locus (e.g., 3p13-14 for renal cell carcinoma, 12p for colorectal carcinoma, and 14q12 for T-cell leukemia), b) The genetic change may be inherited or may occur in the target tissue de novo. In the latter case, the normal somatic cells show no

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chromosomal abnormalities. In cases where constitutional abnormalities with specific breakpoints exist, the carriers are predisposed to developing specific neoplasms.

Constitutional chromosome aberration means that the individual zygote starts with a genetic abnormality. However, chromosome aberrations may occur in postzy- gotic stages, resulting in mosaicism of various propor- tions. If a critical aberration occurs in only a small fraction of the normal cells, and if this aberration occurs in the target tissue, the probability of neoplastic conversion will increase dramatically. Thus, in karyolog- ic examination of cultured lymphocytes or fibroblasts, finding occasional cells with special aberrations should be considered a significant discovery. For example, Motegi 131} observed a low percentage of 13q deletion in lymphocytes of a retinoblastoma patient.

Chromosome Instability and Genetic Predispo.sition to Cancer

Specific congenital mutations and chromosome aberrations are excellent examples to support the concept of genetic etiology of neoplasia; but such cases are comparatively rare in occurrence, whereas cancer as a whole prevails worldwide. Thus, a different mechanism or mechanisms of carcinogenesis, such as a high mutation rate, either by genetic defect or by exposure to environmental carcinogens, must be considered. When the rate of mutations and chromosome damage increases, there will be more chance to achieve the combination that favors specific neoplastic transformation. Although methods are available to estimate mutation rates of mammalian somatic cells, they are laborious and expensive for routine use. On the other hand, the frequency of chromosome aberrations can be used to estimate genetic instability or responses to environmental mutagens or both, because chromosome aberrations can be microscopically enumerated in either metaphase figures or prematurely condensed chromosomes of interphase.

For human subjects, cultured lymphocytes and dermal fibroblasts are ideal materials for cytogenetic studies, because the cultures are easy to initiate and have a high rate of success. Cytogenetic recordings of metaphases of blood cultures from numerous normal individuals showed that the frequency of spontaneous chromosome aberrations Iboth chromatid and chromosome types} is usually low (0 to 3% metaphases with aberrations}; but in several of the so-called chromosome breakage syndromes, as high as 50% of the cells may exhibit spontaneous chromosome aberrations.

There are several well-known chromosome-breakage syndromes. These syndromes have been intensively investigated because they exhibit two important characteristics: predisposition to cancer and high frequencies of spontaneous chromosome aberrations. These syndromes support the concept that genetic instability (as detected by chromosome fragility} and neoplasia are related phenomena or, more specifically, that genetic instability

may increase the probability of oncogenesis. I can summarize only briefly the findings pertinent to the present discussion; for a review of the entire field, readers should consult with the book edited by German ~32).

Bloom's syndrome (BSL Bloom's syndrome, which resembles lupus erythematosus in some respects, was first described in 1954 ~33}. The patients usually have short stature and sun-sensitive telangiectatic erythema. The disease has an autosomally recessive mode of inheritance t34}. An abnormally high proportion of patients with this syndrome develop malignancies, many early in life. The neoplasms are not confined to one tissue, but leukemias (acute lymphocytic and nonlym- phocytic} seem to be most common {35,36}.

Cytologically, the Bloom syndrome has several unusual characteristics: a} A much greater-than-normal frequency of spontaneous chromosome aberrations {37}. Usually 5 to 15~ of metaphases exhibit a variety of chromosome or chromatid aberrations or both in cultured lymphocytes, fibroblasts, bone marrow aspirates, and lymphoid cell lines; b} a certain proportion of metaphases with symmetrical chromatid exchanges between homologous chromosomes; and c} an extremely high rate of spontaneous sister chromatid exchanges (38}.

Ataxia telangiectasia (ATL Ataxia telangiectasia, first described by Syllaba and Henner 139}, is a member of genetically determined immunodeficiency syndromes. It is characterized by a decrease or absence of immunoglob- ulin IgA, acquired agammaglobulinemia, immunodeficiency accompanied by ataxia, and telangiectasia of the conjunctiva and the skin. Like BS, AT patients also seem to be predisposed to neoplasia ~40}. The prevalent types of neoplasms of AT patients are the non-Hodgkin's and Hodgkin's lymphomas. However, leukemias and carcinomas have also been found.

Hecht et al. t41} reported chromosome instability in AT. In numerous subsequent investigations, chromosome aberrations ~both chromatid and chromosome types} have been found as consistent cytogenetic features of cultured lymphocytes and fibroblasts. In a number of cases, clones with rearranged chromosomes persisted, especially rearrangements involving N 7 and N 14.

Fanconi's anemia (FA). This syndrome ~42} is characterized by a number of physical and mental deformities, early development of aplastic anemia, and neoplasia. The neoplasms include leukemias, squamous cell carcinomas of various tissues, and hepatomas. Chromosome instability can be found in cultured lymphocytes, dermal fibroblasts, lymphoid cell lines, and direct bone marrow aspirates ~43-45L Up to 52% of metaphases may exhibit chromosome aberrations t46}.

Werner's syndrome (IVS). In many respects, WS ~47} may be considered a syndrome of accelerated aging. The patients usually have short stature, premature graying of hair, early development of cataract, malignancies of connective and epithelial tissues, and leukemias. Skin fihroblasts and lymphocytes of WS patients show a high frequency of variable but stable translocation 148-50}. This phenomenon is referred to as variegated transloca-


tion mosaicism. In cultured lymphocytes an increase in chromosome breakage was reported by Nordenson (51} in four WS patients.

Xeroderma pigmentosum (XP). Traditionally, XP has been included in the chromosome breakage syndromes, but XP is not abnormally high in frequency of spontaneous chromosome aberrations. Cytogenetic findings of XP will be summarized in a later section.

.4 unique case of chromosome instability and carcinogenesis in utero. A unique case described by Ledbetter et al. 152} emphatically supports the notion that chromosome instability is a predisposing factor to oncogenesis. A family had three children, two girls and a boy. Both girls were born with mult iple malignancies and other developmental anomalies. Cultured lymphocytes of both girls and fibroblasts of the second exhibited numerous random chromosome rearrangements, including clones with the same translocations. In addition to chromosome- type aberrations, cultured lymphocytes of both girls showed extensive chromatid breaks and chromatid exchanges. Chromatid breaks per cell of the first and the second girl averaged 0.92 and 0.91, respectively. They were higher than those in most chromosome breakage syndromes. Both parents are cytogenetically normal.

This case probably represents the extreme end of the chromosome instability spectrum, as malignancy development in utero is an unusual occurrence and the abnormally high frequency of chromosome instability is also unprecedented. I t is not a surprise to find that the dermal fibroblast line succumbed after only a few passages.

Others. A number of investigators reported that, in some human diseases other than the chromosome breakage syndromes, spontaneous chromosome aberrations occur somewhat more frequently than they do in normal control persons, but the difference is relatively subtle t53-55L Using spontaneous chromosome aberration rates as an indicator of genetic instability has several disadvantages, a} The frequency of spontaneous chromosome breakage is usually not high in most persons, including many cancer patients. To obtain statistically acceptable data, a minimum of 200 metaphases should be scored. For studying a large number of individuals, this approach is impractical, b} The spontaneous chromosome breakage rates, expressed as mean breaks per cell tb/c}, of healthy control individuals vary from individual to individual and from sample to sample. Variation among individuals with the same syndrome is equally great, so that overlap between persons with mild degrees of genetic instability and normal individuals always occurs. Again, this causes difficulty in stat ist ical evaluation, c} Hereditary defective DNA repair mechanisms, exemplified by xeroderma pigmentosum, cannot be demonstrated by recording spontaneous chromosome breakages because cells must be challenged with a mutagen or mutagens to reveal such a phenotype. For these reasons, it is felt that mutagen sensitivity experiments may be more revealing for measuring genetic instability than spontaneous aberrations alone. In conducting mutagen sensitivity experiments, control

samples must be taken in any case. Therefore, the rates of spontaneous chromosome breakage can also be determined in these experiments.

The distribution of spontaneous chromosome lesions has not been exhaustively analyzed in terms of their locations, but superficially it seems to he random. The exception is found in Bloom's syndrome, in whose cells exchanges between homologous chromosomes have been frequently found to be at the same loci. If a high rate of chromosome breakage is etiologically associated with cancer init iation, randomly distributed chromosome breakage is a highly inefficient method because it represents a hit-and-miss approach. However, if chromosome lesions are nonrandom, and if the breakage loci happen to be those that are vitally linked to tumorigenesis, then the probability of tumorigenesis would increase drastically in the target tissue. For example, if rearrangements involving the lq21 band indeed initiate breast cancer, as advocated by Cruciger et al. t15}, then a nonrandom breakage at that locus should greatly increase the incidence of breast cancer in individuals who carry such a trait. The discovery of fragile sites in some human congenital syndromes (e.g., fragile X} has stimulated great interest among cytogeneticists. For detailed accounts the readers should consult the excellent book recently published (56}.

I t is not the purpose of this article to review the findings on fragile sites unless the fragile sites are related to oncologic problems. The thought that heritable fragile sites may contribute to cancer, especially familial cancers, is most attractive 157,58~. Nevertheless, thus far there has been no concrete evidence to link specific fragile sites with specific cancers. The difficulty in this endeavor is our lack of critical information regarding specific chromosome alterations of most neoplasms. Once specific chromosomal lesions in many tumors are known, it should be possible to find out whether fragile sites in these specific loci are expressed, particularly in familial tumors.

Mutagen Hypersensitivity

The relationship between high frequency of spontaneous chromosome breakage and high incidence of cancer offers a clue to the cause of carcinogenesis; but chromosome breakage syndromes, like specific congenital chromosome aberrations, are not common enough to account for the abundance of cancer in the human population. Moreover, it has been established that cancer can be caused by environmental factors. Voluminous reports in the scientific literature demonstrate that most carcinogens are genotoxic. However, different persons seem to respond differently to the same or similar environment. Setlow (59), Glickman ~60~, and other biologists voiced the opinion that differences in DNA repair mechanisms as responses to genotoxic effects of mutagens may play a role in an individual's susceptibility or resistance to carcinogens.

The best example of hereditary hypersensitivity to an environmental mutagen and subsequent cancer develop-

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ment is xeroderma pigmentosum. The patients show increased susceptibility to sunlight-induced cutaneous damage, resulting in acute sun sensitivity, abnormal pigmentation, atrophy of the skin, and cutaneous tumors, both benign and malignant.

Most investigators include XP in the chromosome breakage syndromes. In general, the frequency of spontaneous chromosome breakage is not high in XP cells {61~. However, after irradiation with ultraviolet light, Parrington et al. {62) found, in cells of three patients, that the frequency of chromosome aberrations was significantly higher than cells of normal individuals similarly irradiated. Similarly, UV-induced mutation frequency is also higher in XP cells {63). Thus, XP represents a hereditary disorder that is hypersensitive to a type of mutagen {UV) whose biochemical mechanism is known. In this respect, XP differs from the chromosome breakage syndromes in that the latter exhibit an elevated frequency of spontaneous chromosome aberrations, whereas XP cells do not.

I t has been well known that UV induces formation of pyrimidine dimers of DNA, and the cells need excision repair enzymes to repair the damage. The level of repair enzymes is deficient in XP patients {64,65L The specificity of XP hypersensitivity to UV light as an expression of specific repair deficiency is complemented by findings of lack of hypersensitivity of these cells to ionizing radiation {66,67) and to several DNA crosslinking agents ~68-70).

If genetic defects can occur in excision repair as exemplified in XP individuals, similar defects or deficiencies may also exist in other enzymes employed for DNA repair. Because different mutagens have different mechanisms for inducing DNA damage, it is not illogical to infer that different genetic defects may exist among individuals in response to one type of DNA damage but not to all types. Indeed, it has been known that cells of AT patients are sensitive to ionizing radiation {71,72) and to bleomycin (73; but are not hypersensitive to UV light (74,75). The cells of FA patients are sensitive to a number of alkylating agents {76-80L Bloom's syndrome is not known to be hypersensitive to either UV light or ionizing radiation. Heddle et al. {81) have presented a detailed synopsis and discussion of this area of study. The data summarized above suggest that a) spontaneous chromosome instability and mutagen hypersensitivity are separate phenomena, and b) persons with one syndrome may be hypersenstive to one mutagen but not to another.

It should be emphasized that XP, AT, and FA represent the extreme end of the distribution spectrum of mutagen susceptibility. The overwhelming majority of individuals in the human population should not have such severely defective DNA repair mechanisms. However, a degree of mutagen susceptibility may exist in some persons because of mild imperfections of DNA repair systems. Conceivably, such defects may cover a wide range from slightly inefficient to moderately defective. If so, persons with mild degrees of mutagen sensitivity may be more likely to accumulate mutations and chromosome aberrations in their cells when they are exposed to environmen-

tal mutagens and are consequently more susceptible to carcinogenesis.

To demonstrate the variability in mutagen sensitivity among normal persons, a routine assay system must be developed. Theoretically, this assay system should include: aj a simple routine protocol by which investigators and technicians can complete the assay of each sample during usual work hours, b) procurement of test cells should be the least objectionable to donors while yielding a high success rate, c) the feasibility of testing large numbers of normal persons and cancer patients, and dj the opportunity to test as many mutagens with different mechanisms as practicable.

Because of our interest in genetic predisposition to neoplasia, members of my laboratory began several years ago to explore the possibility of differential susceptibility to mutagens among human individuals. At one time we studied the clastogenic properties of gentian violet and used this stain as our test agent. Preliminary data {82) encouraged us to proceed in this direction, but we found that gentian violet was not a suitable clastogen.

To replace gentian violet, we tested a number of agents: ionizing radiation, bleomycin, the alkylating agents triethylenemelamine {TEML MNNG, mitomycin C, actin- omycin D, and others. We finally decided to use the radiomimetic antibiotic bleomycin as the principal test compound. With a short-term exposure to bleomyein, metaphase chromosomes showed an increased frequency of chromatid breaks with apparent random distribution along the chromosomes.

Using cultures of peripheral blood samples, we tested concentrations of bleomycin and treatment durations to obtain an assay protocol at which an average of 0.50 chromatid breaks/cell could be induced in the lymphocytes of normal individuals. We decided that 30 ~g/ml of bleomycin for a treatment time of 5 h before cell harvest was acceptable as our routine procedure. Metaphases arrested with a 1-h Colcemid treatment before harvest should have represented cells in the mid-late S phase when bleomycin was introduced. There should be sufficient time to allow for DNA repair.

Our earlier data {83,84) from testing of 100 normal individuals and 75 patients with various malignancies, can be summarized as follows: a) Responses to bleomycin treatment varied widely among normal individuals. The mean chromatid b /c rates ranged from 0.10 to more than 2.00, but only 12% of control individuals had b/c rates higher than 1.0 and approximately 22% had b/c rates higher than 0.80. b) The reverse distribution was found in cancer patients, viz, approximately 60% of these responded with b /c rates above 1.0.

These preliminary data suggested that, among normal individuals, cellular sensitivity to bleomycin varies widely, from highly resistant to highly susceptible, presumably due to repair capacity. Thus, in an environment with a high mutagen content, hypersensitive {susceptible) individuals may acquire more mutations and chromosomal damage than the resistant ones and are more liable to develop cancer. Unfortunately, direct proof cannot be obtained, because one must wait 20 to 30


yr to verify this assumption, provided that all these tested individuals can be traced at that time. The tests on cancer patients provided indirect evidence, i.e. if the hypersensitive persons are more prone to develop cancer, then we should be able to find more hypersensitive individuals by testing cells of cancer patients because they already have cancer. The high proportion of hypersensitive cases among the cancer patients assayed seemed to support this notion.

Since our previous report (84), we have continued to analyze, using our standard protocol, both normal persons and cancer patients. We can now classify a few types of cancer, including colorectal carcinoma, lung carcinomas, and breast cancer in which 10 or more patients have been studied, to determine the distribution patterns of induced chromatid breakage t85). The distribution pattern of 172 normal persons remained heterogeneous and did not significantly differ from that of the previous sample of 100; but a pattern seemed to emerge among cancer patients, viz, a high proportion of patients with colon and lung carcinomas was found to be hypersensitive to bleomycin-induced chromosome damage, whereas a low sensitivity was recorded in breast cancer patients. In fact, the distribution of bleomycin-response classes in breast cancer patients was almost identical to that of the control persons. I t seems that mutagen hypersensitivity may play a significant role in carcinogenesis in organs directly in contact with the environment, such as the respiratory system, the digestive system, and the integument. For internal organs, other mechanisms of carcinogenesis, such as viruses, hormones, etc., may be more important than mutagen sensitivity, because many genotoxic agents (except ionizing radiationM are metabolized before reaching these tissues. By the same token, it is highly unlikely that mutagen sensitivity is a decisive factor in most childhood cancers, because children do not have sufficient exposure to mutagens except in XP cases. Hereditary lesions probably exist in the majority of childhood tumors, but most such lesions have not yet been identified.

Mutagen hypersensitivity may explay why some cigarette smokers develop lung cancers while others, with an equal history of smoking, do not. Extending this line of reasoning, we would expect that the great majority of smokers reaching advanced age with no sign of lung cancer belong to the group more or less resistant to mutagens. From a limited number of volunteer old smokers thus far assayed, our data seem to support this hypothesis.

Theoretical and Technical Considerations in Mutagen-Sensitivity Test

In the early phase of investigations on mutagen sensitivity in patients with special syndromes, it was necessary to test a number of mutagens with a variety of protocols. Therefore, the data of various investigators cannot be directly compared. However, to find the distribution pattern of mutagen sensitivity and to identify the possible at-risk fraction among normal individuals, several basic and technical problems must be resolved so that procedures and recording methods can be standardized.

Although this article is really not the place to present technical details, we attempt to describe and to discuss many aspects of these to elicit discussion and criticism with the hope that some agreement among cytogeneticists can be reached and a standardized procedure can be established.

DNA repair and mutagen hypersensitivity. In all cytogenetic tests of comparative mutagen sensitivity, we assume that at a given dose under prescribed test conditions, a given mutagen would induce a more or less equal number of DNA lesions and that the differential response is a reflection of the DNA repair capability of the individuals. In XP, this conclusion is certainly valid, because biochemical data support the cytogenetic data. Such correlated information, however, is not available for other mutagens. In most test procedures, the drug- treatment schedule ~for example 24 h) is too long, because too many factors can enter the picture. With a shorter exposure time, cytogenetic methods can at least offer some indirect information on a person's repair capability.

In the assay system we use, viz, bleomycin treatment for 5 h before cell harvest, we conducted a series of experiments using combined treatment with bleomycin and aphidicolin. Aphidicolin has been known to inhibit the activity of DNA polymerase a (86), which is involved in DNA replication as well as in DNA repair.

Aphidicolin itself is a clastogen, but available data indicate that it causes chromosome breakage only in ceils in the S phase. In cultured lymphocytes, only a small fraction (less than 10%) of cells originally in the late S phase can reach metaphase within 2 h. When aphidicolin alone was used to treat the cultured lymphocytes for 2 h, only a low rate of chromatid breaks was recorded (87). Using the same treatment duration with bleomycin, a higher rate of chromatid breaks was registered. If the two agents act on separate mechanisms, the rate of chromatid breaks should be the sum of the two when the cells are treated with both agents simultaneously. The actual results, from a large number of experiments, showed that the two agents had a synergistic effect, with chromatid break rate three to five times the expected additive value tFig. 1L Moreover, breakage rates of the combination treatment were invariably high regardless of the variability recorded in the 5-h bleomycin-treated series. These data offer the following conclusions: a) Under a given condition Itype of culture, dose of drug, duration of treatment), bleomycin induces approximately the same number of DNA lesions, b) DNA polymerase a is definitely involved in repairing bleomycin-induced DNA damage, and c) the rate of observed chromosome breaks in the final harvest depends largely on the DNA repair capability of the individual, i.e. a high rate of chromatid breakage suggests an inferior repair capability.

Although Bender (88) did not investigate the problem of differential susceptibility to x-ray among human individuals, his data on x-ray and aphidicolin cotreatment were almost identical to ours in demonstrating an obvious synergistic reaction. Bender's data also suggest that DNA polymerase ct is involved in repair of x-ray-induced chromosome breakage.

598 HSU

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I

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~ 1 7 6

~ o ~ �9 �9

0.5 ". " �9 g

~

~ 1 7 6

o:5 l:O l:~ 2:o 2:5 BLE:OMYCIN- ,5 hr

FIG. 1. Coordinated plot of chromatid breaks per cell induced by bleomycin and T E n from cultured lymphocytes of 46 individuals. Each blood sample was treated with the two agents separately for 5 h.

I t is known that mammalian DNA polymerase a is involved in both DNA replication and DNA repair, whereas DNA polymerase /3 is used exclusively for DNA repair. The only chemicals known to inhibit DNA polymerase /3 are the dideoxynucleoside triphosphates (89), but no cytogenetic data on these inhibitors are available. One reason may be that these chemicals are impermeable through the plasma membrane. However, we have prel iminary information to suggest that polymerase /3 may also be involved in repair of bleomycin-induced chromosome damage, although not as strongly as aphidicolin.

Based on a similar interpretation but without the use of a repair inhibitor, Parshad et al. (90~ x-irradiated cultured fibroblasts from patients with cancer-prone syndromes and from normal control persons in the G2 phase and compared the frequencies of induced chromatid damage. These authors also concluded that the differences between the cancer-prone syndromes and normal controls suggested a difference in DNA repair capabilities. Inasmuch as most of the syndromes are hereditary, implicit is the indication that differential DNA repair capabilities are genetically determined.

Multiple mutagen tests. As mentioned previously, mutagen sensitivity tests should not be limited to one agent, because the mechanisms of DNA damage and repair are different. We have tested 46 blood samples to compare the frequencies of chromatid breaks induced by bleomcyin and by the alkylating agent TEM. In this set of experiments, the dose of bleomycin was the same as our standard (30 tag/ml}, whereas that of T E M was 3 X 10 -7 M. The treatment time was 5 h for both drugs. The coordinated plot (Fig. 21 shows a positive, though imperfect, correlation between the two. I t seems that, with the given concentrations, T E M induced more chromatid breaks than bleomycin. Nevertheless, this information suggests that cytogenetic response to bleo-

mycin alone may be sufficient to represent both radiomimetic chemicals and alkylating agents.

On the other hand, we have already learned that persons who are genetically hypersensitive to UV light are not necessarily hypersensitive to ionizing radiation, and vice versa. Thus, in testing genetic sensitivity for skin cancers, at least two agents, ionizing radiation (or a radiomimetic chemicaB and UV light (or a UV-mimetic chemicall should be employed. Skin encounters all types of genotoxic agents in the environment, whereas UV light is not expected to cause severe DNA damage in cells of internal organs. I t should be possible, after refining the procedures, to determine which persons are more likely to develop skin cancers. Normal persons may be sensitive to only one agent, both agents, or neither.

Genetic basis. Although data are available to demonstrate that the chromosome breakage syndromes have a hereditary basis, no genetic information has been available on mutagen susceptibility in normal individuals. This question must be answered by pedigree analysis and particularly by testing identical twins. Our limited data on these aspects are not sufficient to warrant a conclusion.

Schedules for drug treatment. In conducting assays using large numbers of human subjects, stimulated lymphocytes in peripheral blood cultures are the only sensible test cells. All cytogeneticists know the procedure. In our laboratory, the routine system uses 1 ml of

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FIG. 2. Chromatid breaks per cell in cultured lymphocytes treated with bleomycin ~2 and 5 h), aphidicolin (2 ht, and combination of bleomycin and aphidicolin (2 hh Note variations in response to bleomycin t5 h) and the generally high responses to the combination treatment.


blood and 9 ml of R P M I 1640 medium supplemented with 15% fetal bovine serum plus phytohemagglutinin. The cultures are incubated for 72 h. Before cell harvest, a 1-h colcemid treatment is given to each culture to arrest metaphases. Conventional air-drying preparations are then made for Giemsa staining without banding.

There are two major treatment schedules for lympho- cyte cultures, treating the cells in G0-G, phases or in S-G2 phases. For treating cells in the Go phase, the drugs may be added to the cultures from 0 to 8 h after the initiation of cultures. The cells are then washed and reincubated in control medium for 48 to 72 h. Chromosome-type aberrations will be the principal abnormalities for quantitative assessment. For testing cells in G, phase, pulse treatment may be performed 12 to 18 h after initiation of blood cultures. In some types of experiments, especially measuring unscheduled DNA synthesis tUDS~ as an indicator of DNA damage and repair, Go-G, cells are preferred. Because no DNA replication has started in G, cells, incorporation of labeled DNA precursors will exclusively indicate UDS. However, many chemical mutagens that have affinity with DNA may remain in the G, nuclei and subsequently induce chromatid-type aberrations even if excess mutagen molecules are thought to be removed from the medium.

Treating cells in S-G2 phase with mutagens will induce chromatid-type aberrations. Treatment time (either pulse treatment or continuous treatment) may vary from less than 1 to 8 h. The concentration of the mutagen used should depend on the desired effect and the potency of the mutagen. For est imating induced chromosome damage, it is undesirable to examine metaphases with too many lesions or too few lesions. The duration should be long enough to allow repair mechanisms to operate. A wide range of concentrations of each mutagen should be tested and the one that would give a moderate number of chromatid breaks, e.g., an average of 0.50 breaks/cell , should be chosen.

We do not infer that the 5-h bleomycin-treatment (30 /~g/ml) schedule that we used should be adopted by all investigators and for all mutagens. However, because we have already accumulated a considerable amount of data using this routine assay, we urge other investigators to include this protocol for comparison.

Ideally, each individual should be assayed four times using blood samples taken at different intervals to cover fluctuation. Such a practice is not always feasible, especially when a laboratory deals with large numbers of individuals. However, whenever possible, an average value from two samples of the same subject is desirable. I t is also desirable to obtain consent of donors for two or more blood samples before taking the first sample. I t avoids suspicion that something may be wrong when the donor is asked for an additional sample several months later.

Recording of chromatid lesions. Many cytogeneticists working in the area of genotoxicity prefer to use sister chromatid exchanges (SCE) as endpoints of measuring genetic response to mutagens, mainly because reading SCE is more objective than reading chromosome aberrations to which some judgment must be exercised.

Because of ambiguity in some situations and because of lack of agreement in identifying chromosome lesions, there have been discrepancies among data collected by different investigators. However, such discrepancies can be minimized if these obstacles are removed, especially if definitions for each type of lesion can be standardized.

I t is relatively straightforward to record chromosome- type aberrations, such as acentric fragments, ring chromosomes, dicentrices, and obvious markers. Inex- perienced observers may occasionally confuse a chromosome that has overlapping chromatids with a dicentric, but such an error is easy to correct. The real problem is in recording chromatid lesions. Metaphase chromosomes may exhibit attenuated areas that are not obvious chromatid breaks. Most cytogeneticists call such subtle lesions "chromatid gaps," but there are no objective definitions to distinguish chromatid gaps from chromatid breaks, because the lesions (achromatic regionM represent a continuous gradation of length, from barely perceptible weakenings to obvious breaks. Because of these difficulties, an ad hoc committee t91) proposed an arbitrary method to define chromatid gaps and chromatid breaks as follows: When the length of the achromatic region is equal to or shorter than the width of the chromatid, the lesion is called a gap; and when the achromatic segment is longer than the width of the chromatid, the lesion is called a break.

This set of definitions has several drawbacks, but at least there is some objectivity for recording when this system is followed. Most likely, recording a chromatid break with this method means that the chromatid is broken. I t is highly possible that some of the lesions classified as chromatid gaps also represent broken chromatids, but such a question cannot be answered with certainty. If the so-called "chromosome core" revealed by silver staining is a reliable indicator of chromatid intactness, then the majority of chromatid gaps do show discontinuity of the chromatids (92). Nevertheless, for estimating mutagen sensitivity, this question is not of vital importance as long as one realizes that some ambiguity is unavoidable in classifying chromatid lesions. My strong objection is to the set of definitions proposed in the International System of Chromosome Nomencla ture t ISCNt 193), which recommends the use of chromatid break only for the type in which the chromatid fragment is displaced at the other side of the intact sister chromatid. If the fragment is still aligned with the sister chromatid at its original side, the lesion is called a chromatid gap no matter how wide the lesion may be. I believe that the cytogeneticists who originally proposed the term "chromatid break" meant that a chromatid was broken, whether the fragment was displaced or not. The ISCN nomenclature system, which completely ignores this concept, may be convenient for scoring purposes, but the committee should have devised a new set of terms instead of adopting the existing terms and changing their meanings. Using the ISCN method, many chromatid gaps represent outright broken chromatids. Therefore, cytogenetic data collected using the ISCN system cannot be compared with those using the classic system.

600 gsu

An example from my own laboratory illustrates discrepancies in recording chromatid lesions when no criteria for classifying the lesions were set before data collection. In our first attempt to analyze mutagen susceptibility among human subjects, beginning in 1977 (82), my colleague Dr. William Au performed all microscopic recordings. His data on untreated lymphocytes of 14 control indivudals (normal, healthy persons with no family history of cancer) showed a mean value of 0.12 lesions/metaphase. Individual samples reached as high as 0.36, which exceeded some of those of the chromosomes breakage syndromes. Because of our inexperience in working with human chromosomes at that time and because of our concentration on testing cancer patients, the control chromatid lesion values simply did not occur to us as being alarmingly high. We suspect, in retrospect, that several factors might have contributed to the abnormally high frequencies: a} Both prometaphases and metaphases might have been used for recording. The chromosomes of prometaphase, especially early prometaphase, often show more gappy or even beaded morphology than those at full metaphase. Some of the lesions recorded in that work might indeed be false. b) C-band regions of chromosomes 1, 9, and 16 are frequently lightly stained and may be miscontrued as chromatid gaps. c) Understained preparations tend to show more gappy chromosomes than properly stained preparations, whereas overstained preparations tend to obliterate minor chromatid lesions, d~ Most importantly, we did not have an agreement regarding the definitions of chromosome breaks and gaps. However, the faulty control data in that report did not seriously affect the major purpose of that work, i.e. to find whether there was mutagen hypersensitivity among cancer patients.

In subsequent investigations on chromosome instability in human subjects, several investigators in our laboratory made an agreement on criteria of recording chromatid lesions by eliminating probable errors and misjudgments and by stressing a conservative approach, viz, ambiguous lesions were disregarded. We adopted the classic set of definitions mentioned previously ~88) and used two additional criteria: a) If a lesion seemed to be in the category of chromatid break but a thin chromatid strand was present in the achromatic zone, the lesion was still classified as chromatic gap. b) A chromatic gap by definition with its intact sister bent at the lesion site was recorded as a chromatid break.

In all of our exercises, only chromatid breaks were recorded, and chromatic gaps were disregarded. This simplified procedure may be cri t icized by other cytogeneticists, but we found that, for the purpose of measuring bleomycin sensitivity, our method greatly increased efficiency and probably accuracy as well. Using this procedure, the mean value of chromatid b /c from nearly 200 normal individuals was approximately 0.02 trange 0.00 to 0.08L

In bleomycin-treated blood samples, our impression was that chromatid gaps were usually not as frequent as chromatid breaks; but we did not perform a quantitative recording to compare the frequencies of the two types of

lesions in the same sample. To determine whether excluding or including chromatid gaps in the recording system makes a substantial difference in the outcome, we compared data from 10 sets (5 normal subjects and 5 cancer patientsj of cultured lymphocytes selected only for their abundance of mitotic figures and quality of spreads. Each set consisted of two samples: untreated and bleomycin-treated. From each sample, 100 full metaphase figures were scored for both chromatid breaks and gaps. The results are summarized in Table I.

An inspection of Table 1 reveals that in the control blood samples, the frequencies of spontaneous chromatid breaks are very low, so that the frequencies of spontaneous chromatid gaps become proportionately significant. On the other hand, in the bleomycin-treated series, despite the increase in chromatid gaps in all samples, the increase in chromatid breaks overshadows that of the chromatid gaps. Statistical analyses confirm such an impression, i.e. the data suggest that in recording chromosome response to such mutagens as bleomycin, disregarding chromatid gaps does not significantly alter the conclusion.

Other consideration. A couple of additional technical points, the use of coded slides and the number of cells scored per sample may be worth discussion in connection with mutagen sensitivity investigations.

Theoretically, coded slides should be used in all cases to avoid possible bias in recording. In our laboratory, we always use the culture number as a code. However, under special circumstances, such as when a report must be made on an individual or when a preparation to be read represents the only sample of that period, prior knowledge of the sample is unavoidable. In special cases, such as induction of fragile sites or other experiments in addition to the routine experiments on drug response, it is necessary to know what to read beforehand. In clinical

TABLE 1

RE CO RD IN G S ON C H R O M A T I D b/c , C H R O M A T I D g/c, AND T H E I R COMBINATION (bg/c~ IN

10 BLOOD CULTURE SAMPLES ~

Untreated Bleomycin-treated

Culture No. b/c g/c bg/c b/c g/c bg/c

2465 0.00 0.00 0.00 0.70 0.34 1.04 2505 0.00 0.04 0.04 0.62 0.15 0.77 2514 0.02 0.05 0.07 0.66 0.30 0.96 2788 0.02 0.06 0.08 0.38 0.16 0.54 2803 0.01 0.04 0.05 0.33 0.14 0.47

2674 0.03 0.03 0.06 0.86 0.20 1.06 2683 0.00 0.02 0.02 0.66 0.19 0.85 2687 0.02 0.03 0.05 0.25 0.17 0.42 2696 0.00 0.04 0.04 0.93 0.35 1.28 2730 0.00 0.02 0.02 0.57 0.28 0.85

~ first 5 samples were from control individuals; the second 5 samples, from cancer patients. Chromosome-type aberrations were not recorded. Each untreated or bleomycin-treated sample consisted of reading of 100 metaphases.


cytogenetics, a similar situation exists t94). We do not think that bias has played a significant role in our data collection. Our previous preconception that all cancer patients should have a higher degree of mutagen sensitivity than control individuals proved to be wrong when breast cancer patients did not respond as expected ~95). This suggests that our recording had no bias.

On the other hand, decoding should be made soon after data collection is completed. Investigators should periodically check progress to decide whether a project is worth pursuing instead of blindly collecting data without knowing what the results indicate.

It goes without saying that the larger the sample, the more accurate the data. Most investigators score 100 cells/sample, while more fastidious ones score 200 cells/sample. However, with very large numbers of samples for survey purposes or for routine determina- tions, even reading 100 metaphases becomes burden- some. In our laboratory, we usually score 50 metaphases/ sample ~84). The correlation coefficient between 50 and 100 cells seems to be good. One should keep in mind that fluctuation does occur, but for samples in the low response range (b/c = 0.60 or lower) or in the high response range (b/c = 1.00 or higher), some fluctuation would not severely affect the conclusion. Therefore, reading 50 metaphases should be considered acceptable. In the borderline zone (b/c between 0.60 and 1.00), it may be advisable to read 100 cells or to assay two blood samples of the same individual.

In practically all treated cultures, one occasionally will find a cell with an uncountable number of chromatid breaks. Routinely, we do not record the number of breaks from such metaphases because one such cell will greatly alter the overall data. Perhaps adding the percentage of cells with chromatid breaks will add value to the estimate.

SUMMARY AND CONCLUSIONS

From studies on cancer genetics, available information suggests the following tentative conclusions:

1. Cancer starts with a genetic change (or changes) from a normal somatic cell, but the changes (mutational events) must be specific in a target tissue cell. In a number of cases, genetic changes can be detected at the chromosome level.

2. Hereditary cancers usually have one genetic lesion already existing prezygotically; therefore only one additonal mutational event is required in the homologous gene to complete the process of neoplastic transformation. In nonhereditary neoplasms, both mutations must occur postzygotically.

3. Individuals with high spontaneous mutation rates (monitored by chromosome breakage rates) are more liable to acquire specific genetic lesions than those with low mutation rates; therefore they are at higher risk to develop neoplasms.

4. Individuals with genetic defects in response to damage induced by mutagens (carcinogens) are more liable to accumulate genetic lesions than those who are more resistant; therefore, they are more liable to develop

cancers. Mutagen sensitivity or resistance is probably genetic expression of DNA repair capabilities.

5. An effective assay method for sensitivity or resistance to mutagens can be developed to analyze the human population to identify the at-risk fraction.

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Supported in part by Research Grants from the John S. Dunn Foundation, Houston and CA-35007 from The Nat ional Cancer Institute.

Documents

Genetic predisposition to cancer with special reference to mutagen sensitivity