[CANCER RESEARCH 54. 582-586, January 15.19q4]

The Involvement of "Tumor Suppressor" p53 in Normal and Chronic Myelogenous

Leukemia Hemopoiesis

S u c a i Bi , 1 F r a n c e s c o L a n z a , 2 a n d J o h n M . G o l d m a n 1

Leukaemia Research Fund Centre for Adult Leukaemia, Department of Haematology, Royal Postgraduate Medical School, London, England

A B S T R A C T

We investigated the expression of p53 in paraformaldehyde-lysine-peri- odate fixed normal and chronic myelogenous leukemia (CML) hemopoi- etic cells with flow cytometry and two monocional antibodies, PAbI801 and the mutant-conformation-associated PAb240. With both antibodies p53 proteins were detected in more than 50% of CD34 § cells and in more than 95% neutrophils but were undetectable in the CD34- myeloid pre- cursors. The expression of a p53 protein reactive with PAb240 was closely associated with CD34§ + cells and with cells in active cell cycle, while the p53 protein recognized by PAbl801 was mainly found in CD34+/ HLA-DR- cells and in cells in the Go/G1 phases of the cell cycle. Treatment of chronic-phase CML cells with p53 antisense oligonucleotides resulted in significantly increased numbers of granulocyte-macrophage colony-form- ing unit colonies in 12 of 17 cases studied. Slightly reduced granulocyte- macrophage colony-forming unit colony numbers were observed in one case and no change in the four others. In eight samples of normal bone marrow cells, treatment with antisense oligonucleotides showed no con- sistent changes in granulocyte-macrophage colony-forming unit numbers. Our data suggest that the expression of the tumor suppressor p53 is involved in the regulation of both normal and CML hemopoiesis and that the inhibition ofp53 expression could modulate the proliferation of CML hemopoietic cells and possibly of normal cells.

I N T R O D U C T I O N

Mutations in the p53 tumor suppressor gene are among the most frequent molecular changes in human oncogenesis (1). Wild-type p53 has been defined as a tumor suppressor gene because of its ability to inhibit transformation (2, 3). Mutations in the p53 gene often result in

an increased steady level of mutant protein (4). Both wild-type and mutant forms of the p53 protein are believed to play an important role in controlling cell proliferation for a number of reasons: wild-type p53 protein can negatively control cell proliferation and act as a tumor suppressor in in vitro transformation assays (2, 3, 5-7); overexpres-

sion of wild-type p53 can cause cell cycle arrest at the G1/S-phase border (5, 8, 9); and introduction of a plasmid expressing antisense p53 RNA can inhibit the proliferation of transformed host cells (10).

The p53 tumor suppressor gene is inactivated in various malignan- cies, including leukemia and lymphoma (1, 11-15). Although consid- erable attention has been focused on the structural changes in p53 and

their biological consequences, the normal function of wild-type p53 in hemopoiesis is still uncertain. For example, it is uncertain whetherp53 is expressed by hemopoietic stem and progenitor cells, although it is expressed in mature blood cells of all lineages (16, 17). p53 expres- sion can be induced in differentiated cells of a myeloid cell line (ML-1) which expresses little or no p53 protein during proliferation at immature stages (16). Expression of wild-type p53 has been linked with apoptosis in a murine myeloid leukemia cell line. Its apoptosis- mediating effect can be counteracted by interleukin 6 (18).

In order to elucidate the exact role of p53 in hemopoiesis, we used

flow cytometry to study the expression pattern of p53 in hemopoietic

Received 7/19/93; accepted 11/15/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Leukaemia Research Fund Centre for Adult Leukaemia, Department of Haematology, Royal Postgraduate Medical School, Du Cane Road, London W12 0NN, England.

2 Present address: Institute of Hematology, University of Ferrara, Ferrara, Italy.

cells and its association with states of cell cycle and differentiation.

The effect of inhibiting p53 expression by antisense oligonucleotides on the proliferation of normal and leukemic hemopoietic cells was also investigated by using the CFU-GM 3 assay.

M A T E R I A L S AND M E T H O D S

Sources of Samples. Normal and pH-positive CML bone marrow aspirates were obtained from normal bone marrow donors and CML patients in chronic phase who underwent bone marrow harvesting for allogeneic or autologous bone marrow transplantation in our department. Informed consent was ob- tained in each case. Light density mononuclear cells were prepared by centri- fuging bone marrow cells over a layer of Percoll of desired density (1.070 and 1.067 g/ml for normal and CML bone marrow, respectively). Neutrophils were also isolated from the buffy coat layer in some of the samples for detection of p53 expression. The isolated light density cells were washed twice in Hanks' balanced salt solution (Flow Laboratories, Irvine, Scotland) before being re- suspended in Hanks' solution or in SFM for analysis of p53 expression and for clonogenic assays, respectively. For negative and positive controls, three leu- kemia cell lines, HL-60, K562 (both p53 negative), and KYO-1 (p53 positive), were used inasmuch as their ability to express p53 has been defined in flow cytometry studies (19, 20).

We used either negative depletion or a FACS for selection of CD34 + cells. For negative depletion, we used a cocktail of 8 monoclonal antibodies, which recognize various differentiation and lineage-associated antigens: CD2; CDllb; CD14; CD15; CD19; CD22; CD42; and glycophorin A (Gly A) (all from DAKO, High Wycombe, England), in conjunction with sheep anti-mouse immunoglobulin-coated magnetic beads (Dynabeads) (Dynal, Oslo, Norway) to remove the CD34- cells. For positive selection, a standard procedure was used to enrich CD34 + cells on a Epics fluorescence-activated cell sorter (Coulter, Hialeah, FL). The purities of CD34 + cells after negative depletion and FACS sorting were 67-92% and >96%, respectively.

Monoclonal Antibodies, Cell Cycle Analysis, and Flow Cytometry. Two monoclonal antibodies, PAb1801 and PAb240 (Oncogene Science, Cambridge, England), were used to detect p53 expression. The antibody PAb1801 recog- nizes an epitope near the NHz-terminal end of the p53 peptide present in all species studied (21); it reacts preferentially with human p53. The antibody PAb240 was originally reported to recognize a common conformational epit- ope closely associated with mutations in the p53 gene (22), but recent findings suggest that it can also react with undenatured wild-type p53 protein in he- mopoietic cells (17, 23).

For the detection of p53 expression by flow cytometry, cells were first fixed at -8~ to -10~ for 5 min in paraformadehyde-lysine-periodate fixation solution. After removal from the fixation solution, cells were washed in phos- phate-buffered saline and incubated with one or another of the p53 mono- clonal antibodies for 30 min at 4~ washed in phosphate-buffered saline, and incubated with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG monoclonal antibody (DAKO, High Wycombe, England) for 20 min at 4~ An irrelevant IgG1 monoclonal antibody was used in parallel as an isotypic control (Oncogene Science, Cambridge, England) before incubation with the conjugated secondary antibody. In some cases, double staining was carried out with phycoerythrin-conjugated CD34 monoclonal antibody (Becton- Dickinson, Oxford, England) and fluorescein isothiocyanate-conjugated PAb240 (Oncogene Science, Cambridge, England) to detect the coexpression of CD34 and p53. Flow cytometric analysis was carried out on a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA).

The assessment of cellular DNA content was made with a flow cytometer (Becton-Dickinson, Mountain View, CA). The cell cycle distribution data were

3 The abbreviations used are: CFU-GM, granulocyte-macrophage colony-forming unit; CML, chronic myelogenous leukemia; FACS, fluorescence-activated cell sorter; SFM, serum-free medium.

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p53 EXPRESSION AND NORMAL/CML HEMOPOIESIS

provided by the Rectangle-Fit (R-FIT) mathematical algorithm of the Facscan/ Cellfit software program. The double discrimination module was used to detect doublets and higher cell aggregates. To determine DNA content, cells were treated with 0.5% Triton X-100 for 3 min and then stained with a propidium iodide solution (50 ~,g/ml). RNase (Sigma, Dorset, England), at a final con- centration of 0.1%, was added to the cell preparation. Bromodeoxyuridine incorporation and a monoclonal antibody against bromodeoxyuridine (Becton- Dickinson, Oxford, England) in conjunction with flow cytometry were used to calculate the size of population of cells in S phase of the cell cycle. The manufacturer's recommendations were followed for labeling cells and detec- tion.

Confocal Microscope. A laser scanning confocal microscope (MRC-600; Bio-Rad, Hercules, CA) was used to study the cellular localization of p53 in purified CD34 + cell stained with p53 and Texas red-conjugated (Vector Labo- ratories, Peterborough, England) or fluorescein isothiocyanate-conjugated (DAKO, High Wycombe, England) secondary antibody.

Antisense Oligonucleotides. Three 18-mer oligonucleotides were synthe- sized on a DNA synthesizer (380B DNA synthesizer; Applied Biosystems, Foster City, CA) by an automated process. The antisense sequences target the region covering the six base pairs immediately before the first codon and the first four coding codons. Sense sequences corresponding to the antisense target region were used as a control. A scrambled antisense oligonucleotide was also used in some experiments. The sequences of the three oligonucleotides were as follows: 5'-CGG CTC CTC CAT GGC AGT-3' (antisense); 5'-ACT GCC ATG GAG GAG CCG-3' (sense); and 5'-AGT GGC CTC CAT CTC CTC CGG-3' (scrambled antisense). In order to increase the nuclease resistance of the oligonucleotides, the 18-mer oligonucleotides were made up of 12-mer of unmodified sequences flanked by 3-mer phosphorothioate sequences at both ends. The synthesized oligonucleotides were ethanol precipitated followed by multiple washings in 70% ethanol (>5 times) for purification. Purified oligo- nucleotides were then dissolved in RPMI 1640 (Flow Laboratories, Irvine, Scotland) and kept at -20~ until use. Since we observed no difference in CFU-GM colony numbers in cells treated either with sense or scrambled antisense sequence oligonucleotides in five cases of CML (data not shown) and in CML cell lines (24), we used only sense oligonucleotides as the control in this study.

Serum-free Medium and CFU-GM Assay. SFM was used for the incu- bation of hemopoietic cells with the oligonucleotides. SFM was prepared according to the method of Salem et al. (25). The basic ingredients were dialyzed bovine serum albumin, cholesterol, insulin, 2-mercaptoethanol, and transferrin (all from Sigma, Dorset, England) mixed in Dulbecco's modified Eagle's minimium essential medium (Gibco, Uxbridge, England). The light density hemopoietic cells were preincubated with oligonucleotides at 37~ for at least 6 h and then plated in standard CFU-GM medium supplemented with corresponding oligonucleotides at a concentration of 5/XM. CFU-GM medium was prepared in a standard manner: 1 • 105 preincubated cells were mixed in 1 ml of Iscove's modified Dulbecco's minimum essential medium (Gibco, Samples Uxbridge, England) containing methylcellulose (1% w/v) (Fluka, Dorset, Eng- land), fetal calf serum (Tissue Culture Service, Buckingham, England) (30% v/v), and 10• concentrated (M~ >10,000 solute retained) supernatant from the 5637 bladder carcinoma cell line (1% v/v). They were plated in 35-mm Petri dishes. Cultures were incubated for 7 days at 37~ in 5% COz humidified atmosphere. Colonies comprising >50 cells were identified and counted with an inverted microscope. All experiments were set up in triplicate.

Statistical Analysis. Paired t-tests or Wilcoxon's matched paired rank tests were used as appropriate. A P value of <0.05 was judged as significant.

RESULTS

Expression of p53 in Hemopoietic Cells. Seventeen samples of normal and CML light density cells were studied for the expression of p53 with two p53 monoclonal antibodies, PAbl801 and PAb240. As

shown in Fig. 1, the expression of p53 was clearly detectable in normal and CML light density cells with flow cytometry. The per- centage of p53-positive cells recognized by PAbl801 was slightly lower than that with PAb240 (Table 1). No positive signal was de- tected in either HL-60 or K562 cells reacted with PAbl801 or PAb240, while more than 90% of KYO-1 cells reacted with PAbl801. These results indicate that the reactivities with PAbl801 and PAb240 were

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Fig. 1. Expression of p53 in CML light density cells detected by flow cytometry. Top, CML light density cells incubated with the isotypic control antibody; Bottom, CML light density cells incubated with PAbl801.

Table 1 The positivity of p53 and CD34 in light density normal and CML bone marrow cells (median with range in parentheses)

PAb240 (%) PAbl801 (%) CD34+ (%)

Chronic phase CML 16.1 12.4 9.2 (13 cases) (9.8-68.7) (3.7-29.5) (4.2-20.2)

Normal BM 13.2 9.4 5.5 (4 cases) (6.0--14.3) (6.2-10.7) (3.2-6.1)

p53 specific. In addition, we found that slightly more CML cells expressed p53 proteins than did normal bone marrow samples. The percentage of CD34 + cells among these light density cells was 5.5% and 9.2% in normal and CML samples, respectively. Of these CD34 + cells, 42% and 57% coexpressed p53 in normal and CML samples,

respectively, as detected by double staining. Using purified CD34 + cells, comparable results were obtained for the coexpression of p53

and CD34: 53-60% of CML CD34 + cells and 56-64% of normal CD34 § cells were p53 positive (Table 2).

To confirm the results with light density cells and to study the

cellular localization of p53, we used a confocal microscope to study the expression of p53 in enriched CD34 + cells in one normal and two CML samples. We found p53 protein (PAb240 reactive) present in a mean of 60% of the CD34 + cells, and the p53 detected was localized

in the nuclei of the cells. The purified CD34 § cells were also used to investigate relation-

ships between the epitopes of p53 protein and (a) expression of the

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p53 EXPRESSION AND NORMAL,'CML HEMOPOIESIS

Table 2 The expression of p53 in purified CD34 + cells (median with range in parentheses)

Samples PAb240 (%) PAbl801 (%)

Chronic-phase CML 60.1 (58.2-62.1) 53.1 (47-65.1) (n = 5)

Normal bone marrow 63.5 (62.6-64.5) 55.9 (55.3-56) (n = 2)

differentiation associated HLA-DR antigen and (b) the cell cycle status. The expression of PAb240-reactive p53 protein was found to be closely associated with a population of more differentiated CD34 § cells, namely HLA-DR § cells, and with cells in active cell cycle, while the p53 proteins recognized by PAbl801 were mainly found in CD34+/HLA-DR - cells and cells in the Go/G~ phase of the cell cycle (Tables 3 and 4).

The myeloid precursor cells, identified on flow cytometer by gating elements that showed a consistent side scatter and CD34 negativity, expressed no detectable p53 protein in 10 of the 12 CML samples. However, in the two cases with extremely highp53 expression in their light density cells (p53 positivities with PAb240 were 54% and 69%, respectively), the myeloid precursor cells also had bright staining with PAb240 but not with PAbl801. The myeloid precursor cells from four normal bone marrow samples had no detectable expression of p53.

Expression of p53 was detected in more than 95% of neutrophils derived from seven CML and three normal bone marrow samples (purities >97% and >90% for normal and CML, respectively).

Specific Inhibition of p53 Expression by Antisense Oligonucleo- tides. Purified CML CD34 + cells were used as target cells for the study of specific inhibition of p53 expression by antisense oligo- nucleotides. Cells were incubated with 5 /ZM antisense oligonucleo- tides in SFM for 6--48 h; the same concentration of sense or scrambled antisense oligonucleotide was used as a control. The expression of p53 by the CD34 § cells before and after incubation with oligonucleotides was compared. As shown in Fig. 2, the expression of p53 was spe- cifically inhibited by incubating cells with antisense oligonucleotides for as short a period as 6 h. Incubation for periods longer than 6 h resulted in more effective inhibition (Fig. 3).

Effect of p53 Antisense Oligonucleotides on the Proliferation of Normal and Chronic Phase CML Hemopoietic Progenitor Cells. Eight normal and 17 CML bone marrow samples were used to study the effect of p53 antisense oligonucleotides on the proliferation of hemopoietic cells. Light density cells were first preincubated with 5 /xM antisense oligonucleotides in SFM for 6 h and then plated with CFU-GM medium which contained 5 /~M antisense oligonucleotides. Sense oligonucleotides were used in parallel as controls in every case. The numbers of day 7 CFU-GM colonies formed by cells treated with antisense oligonucleotide and by cells that had received no oligo-

nucleotide were compared with colony numbers cultured from cells treated with sense oligonucleotides (Fig. 4). We found that sense oligonucleotide-treated normal and CML bone marrow cells generally formed fewer CFU-GM colonies than cells that had received no oli- gonucleotide. The difference in CFU-GM colony numbers between sense oligonucleotide-treated cells and cells that had received no oligonucleotide in CML samples was significant (P = 0.005). In normal bone marrow samples, sense oligonucleotide-treated cells formed an average 9.4% fewer colonies than those formed by cells that had received no oligonucleotide (P = 0.09). These data suggest that these oligonucleotides had nonspecific toxicity on hemopoietic cells. Therefore, the effect of antisense oligonucleotides on prolifera- tion was evaluated by comparing the CFU-GM colony numbers be- tween sense and antisense oligonucleotide-treated cells. Increased numbers of CFU-GM colonies were observed in 12 cases (71% of total cases) of CML samples treated with antisense oligonucleotides. No obvious change in CFU-GM colony numbers was observed in the other four cases, and a slightly reduced CFU-GM colony number was observed in the last case. The overall difference between cells treated with antisense oligonucleotides and cells treated with sense oligo- nucleotides in CML samples was highly significant (P = 0.009), and the overall difference between cells treated with antisense oligo- nucleotide and cells that had received no oligonucleotide was also significant (P = 0.049). In contrast to CML samples, treatment of normal bone marrow cells with p53 antisense oligonucleotides showed either small or reduced numbers of day 7 CFU-GM colonies

Table 3 The association of PAb240- and PAb1801-reactive p53 proteins and the stages of cell cycle in CML CD34 + cells

Percentage of positive cells with

PAb240 PAb 1801

Case Go/Ga S-GJM Go/G1 S-G2/M

1 9 34 30 9 2 22 29 31 16 3 7 44 27 10 4 13 47 36 9

Table 4 The association of p53 epitopes with the positivity (%) of HLA/DR antigen on purified CD34 + cells (mean value • SD)

Samples PAbl801 PAb240

Chronic-phase CML (3 cases) CD34 § HLA-DR + 16.2 • 9.4 64.2 --- 11.1

CD34 + HLA-DR- 61.4 + 17.9 27.4 • 10.0

Normal BM (3 cases) CD34 + HLA-DR- 32.0 • 7.2 49.4 ___ 6.2 CD34 + HLA-DR- 58.6 • 5.1 22.1 --- 4.3

Fig. 2. Expression of p53 (PAb 240 reactive) in CML CD34 + cells before and after treatment with p53 sense or antisense oligonucleotide. Left, CML CD34 + cells before incubation with oligonucleotide; Middle, CML CD34 § cells after 8 h of incubation with sense oligonucleotides; Right, CML CD34 + cells after 8 h of incubation with antisense oligonucleotides.

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p53 EXPRESSION AND NORMAL/CML HEMOPOIESIS

0 ='- 120

100

'~ ao

~ 60

"~ 10

X~" 20

14) 6 8 a . 12

r

r

r

'i 24 48

Duration of incubation (hrs) Fig. 3. Specific inhibition of p53 expression in CD34 § cells by incubating with

antisense and sense oligonucleotides. Only one sample was studied for 6 h of incubation, and at least three samples were studied in other experiments. Bars, SD; C], CD34+ cells before inubation; [~, CD34+ cells incubated in SFM; m, CD34+ cells incubated with sense oligonucleotides; II, CD34+, cells incubated with antisense oligonucleotides.

in most of cases. However, a significant reduction in CFU-GM colony numbers was observed in two cases, as was a significant increase in one case (Fig. 4). The overall difference between CFU-GM colony numbers formed by antisense and sense oligonucleotide-treated nor- mal marrow cells was not significant (P = 0.36).

In addition to the changes in CFU-GM colony numbers, we noted that the colonies formed by antisense oligonucleotide-treated cells were generally larger than those formed by either sense oligonucleotide-treated cells or by cells that had received no oligo- nucleotides. The colonies formed by the sense oligonucleotide-treated cells were generally smaller than those formed by cells that had received no oligonucleotides.

DISCUSSION

Alterations in p53 gene have been found in various human leuke- mias and lymphomas (13-15). Overexpression of wild-type p53 pro- tein can cause cell cycle arrest (5, 8, 9), apoptosis (18), suppression of cell proliferation, and malignant transformation (2, 3, 5-7). Expres- sion of p53 is also involved in the maturation of normal hemopoietic cells (16). However, it is uncertain whether p53 protein plays any role in regulating the proliferation of immature hemopoietic cells. In this IR study, we demonstrated that the expression of p53 protein could be specifically detected in paraformadehyde-lysine-periodate-fixed cells o

"o by flow cytometry. The p53 protein was expressed by both normal and ~ 200 - CML bone marrow stem and progenitor cells. At least 50% of CD34 + cells coexpressed p53, while more differentiated cells such as the CD34- myeloid precursor cells had no detectable p53 protein. Nearly ,he all the mature neutrophils expressed p53. "~ 150

IO Since the cells used for the detection of p53 expression were fixed,

the p53 proteins in these fixed cells were probably denatured. Thus o E L.. reactivity with PAb240, which recognizes both denatured wild-type "-

100 and mutant forms of p53 (22), may not represent the presence of p53 protein with the mutant-conformation-specific epitope. Instead, it may "

E reflect only the total p53 proteins. However, our finding that the same = population of fixed cells had different reactivities with PAbl801 and >, 50

PAb240 (such as the CD34- myeloid precursor cells of the two cases o of CML who had a very high level of PAb240 reactive p53 protein, "~ u which was not recognized by PAbl801) and the fact that different ,,- subpopulations of a group of fixed cells had different reactivities with o 0 PAb1801 and PAb240 (such as the HLA-DR § and HLA-DR- of CD34 + cells) in all cases studied strongly suggest that the p53 protein in these cells is not completely denatured. The denaturation-sensitive epitope of p53 recognized by PAb240 was retained in these fixed cells.

The different reactivities with PAb1801 and PAb240 in these fixed cells may reflect genuine conformational differences of the p53 pro- tein. This speculation is supported by the observations of Rivas et al., who found that the p53 proteins expressed by normal bone marrow blasts were PAb240 positive as detected by immunoprecipitation (17). Our data support their conclusion that the expression of a p53 protein reacting with PAb240 in hemopoietic cells is not the result of muta- tions in the p53 gene.

The finding that the expression of PAb240-reactive p53 protein was closely associated with CD34+/HLA-DR + cells, a group of more differentiated cells than CD34+/HLA-DR - cells, and with cells in the active cell cycle implies that the conformational change of p53 protein may be closely associated with its functional state. It is well known that wild-type p53 protein may be present in two different conforma- tions and the function of the p53 protein in normal cell proliferation is comformation dependent (26-29). It is believed that wild-type p53 protein recognized by PAb240 may transiently lose its tumor suppres- sor and antiproliferative functions and thereby permit cell prolifera- tion. Recent findings that wild-type p53 adopts a "mutant"-like con- formation when bound to DNA further support the notion that the functions of p53, such as its DNA binding activity, are regulated by its conformation (30). The fact that the majority of PAb240-reactive p53 proteins were found in CD34+/HLA-DR + cells and cells in the active cell cycle strongly supports this belief. Moreover, the recent report that more than 97% of CD34+/HLA-DR - cells were found in the Go/G1 phase of the cell cycle and that CD34+/HLA-DR - cells enter- ing into S/Gz/M phases following growth factor induction was ac- companied by the expression of HLA-DR antigen also further sup- ports the assumption that conformational changes in the p53 protein are associated with the state of the cell cycle (31).

The number of CFU-GM colonies formed by cells treated with antisense oligonucleotides was increased in 12 of 17 CML samples, and the colonies were generally larger than control colonies. This suggests that inhibiting p53 expression may promote the proliferation of CML hemopoietic cells. Although we have no definite evidence that p53 is directly involved in the regulation of the proliferation of CML hemopoietic cells, we have observed that inhibition of p53 expression can stimulate cells into the active cell cycle, as demon- strated by the increased expression of the nuclear antigen Ki-67 (32)

~r ~t ~r r

~r

J, ~ * cb

~r 'A"

AS-CML CML-control A$-NBM NBM-control Fig. 4. CFU-GM colony numbers in cells treated with sense and antisense oligonucleo-

tides in comparison with cells that had received no oligonucleotides. AS, antisense oli- gonucleotides; control, cells that had received no oligonucleotides; NBM, normal bone marrow cells (n = 8); CML, chronic phase CML cells (n = 17).

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pS.? EXPRESSION AND NORMAL/CML ttEMOPOIESIS

in p53 ant i sense o l i g o n u c l e o t i d e - t r e a t e d C D 3 4 ~ C M L cel ls (Lanza

and Bi, unpub l i shed obse rva t ions ) ,

In cont ras t to C M L cells , inhibi t ion o f p53 expres s ion had less

e f fec t on normal b o n e m a r r o w cell C F U - G M format ion . A l t h o u g h the

C F U - G M c o l o n y n u m b e r s d i f fe red f rom s a m p l e to sample , 6 3 % of

b o n e m a r r o w samp le s s h o w e d no s igni f icant changes . Ha t z f e ld et al.

repor ted that an t i sense o l i gonuc l eo t i de s agains t p53 had no e f fec t on

c o l o n y fo rma t ion by no rma l h e m o p o i e t i c ce l l s (33); in contras t , w e

did s h o w an e f fec t on normal h e m o p o i e t i c cel ls but m u c h less than

that seen wi th C M L cells .

The f ind ing that inhibi t ing p53 expres s ion increased C F U - G M

c o l o n y fo rma t ion in 7 0 % o f C M L s a m p l e s is cons i s ten t wi th the

obse rva t i on that inhibi t ing exp res s ion o f the r e t inob la s toma gene,

ano ther t u m o r s u p p r e s s o r gene and cell cyc le regulator , cou ld re lease

ear ly h e m o p o i e t i c cel ls f r o m q u i e s c e n c e and s t imula te f ib rob las t cell

d iv i s ion and focus fo rma t ion (33, 34).

We have d e m o n s t r a t e d that the exp res s ion o f p53 dur ing the pro-

l i ferat ion and d i f fe ren t ia t ion o f h e m o p o i e t i c cel ls f o l l o w s a str ict ly

regu la ted pat tern. Thus p53 might be i nvo lved in the regula t ion o f

hemopo ie s i s . S ince p53 can act as a med ia to r o f apop tos i s and a cell

cyc le regulator , its exp re s s ion in i m m a t u r e h e m o p o i e t i c cel ls and

ma tu re b l o o d cel ls m a y not be a co inc idence . In bo th types o f cells ,

apop tos i s is impor tan t to the phys io log i ca l cont ro l m e c h a n i s m . He-

m o p o i e t i c s tem cel ls need to cont ro l their s t em pool s ize p rec i se ly

wh i l e main ta in ing an ac t ive pro l i fe ra t ion and d i f ferent ia t ion process .

The surp lus p ro l i fe ra t ing s tem cel ls mus t be e l imina ted by ac t ive

apop tos i s (35). Ma tu re b l o o d cel ls are des t ined to die at the end o f

their l ife span, p r e s u m a b l y through apoptos is . The re fo re , the func t ion

o f p53 prote in bo th in i m m a t u r e h e m o p o i e t i c s t em cel ls and in mature

b l o o d cel ls m a y be to ac t iva te or med ia t e the p rocess o f apop tos i s and

to regula te cell cyc le status. The d i f ferent c o n f o r m a t i o n a l changes in

p53 in d i f ferent popu la t ions o f cel ls and d i f fe ren t s tages o f the cell

cyc le m a y a lso represen t d i f ferent func t iona l s ta tes o f p53 in d i f ferent

phys io log ica l c i r cums tances . It is un l ike ly that all p53 -pos i t i ve

C D 3 4 § cel ls wil l unde rgo apoptos is . The con fo rma t iona l changes o f

the s a m e prote in in d i f fe ren t s tages o f the cell c y c l e and in d i f ferent

s u b p o p u l a t i o n s o f C D 3 4 § cel ls make it poss ib l e that not all p53-

pos i t ive C D 3 4 § cel ls unde rgo apop tos i s , s ince the c o n f o r m a t i o n s and

func t ions of p53 are regula ted and di f fer in d i f ferent s u b p o p u l a t i o n s o f

ceils.

ACKNOWLEDGMENTS

We are grateful to Dr. Richard Szydlo for helpful advice on the use of statistical methods.

REFERENCES

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2. Finlay, C. A., Hinds, P. W., and I_~vine, A. J. The p53 proto-oncogene can act as a suppressor of transformation. Cell, 57: 1083-1093, 1989.

3. Eliyahu, D., Michlovitz, D., Eliyahu, S., Pinhasi-Kimhi, O., and Oren, M. Wild-type p53 can inhibit oncogene-mediated focus formation. Proc. Natl. Acad. Sci. USA, 86: 8763-8767, 1989.

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in Normal andp53The Involvement of ''Tumor Suppressor''

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