A Mutant P21 Cyclin-Dependent Kinase Inhibitor Isolated from a Burkitt's Lymphoma

[CANCER RESEARCH 55, 1431â€”1435,April 1, 19951 Advances in Brief

A Mutant p21 Cyclin-dependent Kinase Inhibitor Isolated from a Burkitt's Lymphoma

Kishor Bhatia,' Saijun Fan, Gordon Spangler, Michael Weintraub, Patrick M. O'Connor, Jean.Gabriel Judde, and Ian Magrath Lymphoma Biology Section [K. B., G. S., M. W., J-G. J., I. M.J and Laboratory of Molecular Pharmacology (S. F., P. M. 0.1, National Cancer Institute, NIH, Bethesda, Maryland 20892

Abstract p21 gene occur as an alternate mechanism of abrogating p53-mediated growth suppression in BL. The growth arrest mediated bypS3 is caused at least in part by the p53 mediated expression of p21 (p2l@â€•@'1).Since only one-third of primary Materials and Methods Burkitt's lymphomas (BL) demonstrate mutations in the p53 gene, we examined the structural Integrity ofthe p21 coding region by single-strand Cell Culture and Transfection. BL cell lines were derived from lym confomational polymorphism and DNA sequence analysis to determine phoma biopsies at the National Cancer Institute. The BL biopsies were char the extent to which this gene is mutated in BL Of 34 BLs analyzed, a acterized at the molecular level, in particular, chromosomal translocation frequent change (38%) at codon 31 that replaced Ser with Arg was found breakpoints associated with the 8;14 translocation, presence or absence of In 13 samples, 10 of which were from Africa. This change at codon 31 is Epstein-Barr virus, p53 mutations, and presence or absence of mutations also detected in peripheral blood DNA from normal subjects and may thus within the coding region of the c-myc gene were sought. Cells were grown at represent a polymorphism. One BL cell line, DH978, carried a change at 37Â°Cin95% air/5% CO2 in RPM! 1640 containing 15% heat-inactivated fetal codon 63: Phe to Leu. This mutation was heterozygous,and both the bovine serum, 2 mML-glutamine,and 50 @Wmlofpenicillin and streptomycin. wild-type and the mutated p2! mRNA were expressed in the tumor cell All tissue culture products were obtained from BioWhittaker (Walkersville, line. By transfection experiments, the mutant p21 was less efficient In MD) and routinelymonitoredfor Mycoplasmacontamination.Twenty,@gof suppressing clonogenicity than wild-type p21. To our knowledge, this is purified plasmid were transfected in 20 million cells by electroporation. Cells the only mutation described in p21. The availability of this mutant p21 were plated at a density of 2â€”5x10-'cells/ml 24 h after transfection in 24-well should fUrther help In fUnctional studies of p21. plates in media containing hygromycin (500 @Wml).Clonogenicitywas scored between 1 and 2 weeks following transfection. Introduction SSCP Analysis of the p21 Coding Sequences. Lymphoma biopsies from 30 patients collected for routine diagnostic procedures were analyzed. From In addition to a myc/immunoglobulin chromosomal translocation some of the biopsies, cell lines were established. DNA was obtained from the that causes inappropriate c-myc expression, 37% of primary biopsies tumorsamplesusingestablishedprotocols.Southernblot analysisusing the from BL carry mutations in the p53 gene (1, 2). This frequency is c-myc probes spanning the first and third exons were performed as described increased in relapsed tumors (3). p53 mutations in BL may be relevant previously (11). For the PCR-generated SSCP, the initial PCR was performed to pathogenesis, since inactivated p53 is unable to prevent growth using 250 ng of DNA and 10 pmol of each flanking primer. The sequences of arrest and apoptosis and should, therefore, complement neoplastic the primers used for the SSCP were: Waf-1, AGA OGA GGC 0CC ATG TCA proliferation resulting from deregulated c-myc. Alternatively, p53 GAA; Waf-131, TOG OGA CCC TFC AGC CTG CF; Waf-135, TCC AGO mutations may be a component of tumor progression. p5.3 regulates TCCACCTOOGOA;Waf-1.1-3/R,AAGGTAGAGCVF000 CAG0CC; Waf-1.2-5/F, CGA GAC ACC ACF GGA 000 TG; Waf 1.1F, GAG GAG the expression of another growth suppressor gene, p21 (4â€”7).This OCO CCA TOT CAG; Waf 1.2-R,C11@CAGCCT OCT CCC CTO; Waf protein is identical to the previously described proteins, CIP1, CAP2O, RT-5, 0CC OGA Gd 000 COC GGA U; Waf RT-3, GOC CVf TGA p21, and SDI1, and is a potent inhibitor of cyclin-dependent kinases GOC CCT COC OCf; and Waf-140, GAC TCF CAG GOT CGA AA. The (8, 9) which are expressed in G1-S. Thus, p21 may be an important conditions of the PCR reaction and the SSCP analysis have been described mediator of p53-dependent growth suppression. More recently, p21 previously (12). A negative control and a blank PCR (reaction without tem has also been shown to directly bind proliferating cell nuclear antigen plate) was always included in the analysis to confirm the absence of exogenous (10) and halt ongoing DNA synthesis. Consequently, p21-mediated DNA contamination.PCRproductswith aberrantmigrationand several ran growth suppression results from both inhibition of entry into S phase domly chosen PCR products with normal migration were sequenced. Direct and perhaps delayed progression through S phase. If inhibition of the sequencing of the PCR-amplified product was carried out using the Sequenase growth suppressor pathway is important to lymphomagenesis, it kit (USB). Reverse Transcriptase PCR. A eDNA strand was synthesized from ap seemed likely that the two-thirds of primary BLs that lack a p53 proximately 1 @.tgof poIy(A)@ mRNA. The extension reaction was carried out mutation might have lesions in other genes in the growth suppressor using 10 pmol of poly dT primer and 200 pmol of deoxynucleotide triphos pathway. In any event, since p53 growth arrest is mediated by other phates in a reaction buffer containing 50 units of reverse transcriptase. Fol genes, it seemed likely that the same functional effect could be lowing a 30-mm incubation at 37Â°C,one-tenthof the reaction mixture was achieved by mutations in these downstream genes. In the present transferred to a PCR amplification cocktail containing the appropriate primers. studies, therefore, we sought to determine whether mutations in the PCR was carriedout usingstandardprotocols.Eachcycle consistedof 30 s denaturation at 94Â°C,30 s annealing at 58Â°Cand 60 s extension at 72Â°C. Received 12/16/94; accepted 2/17/95. Amplification was carried out for 30 cycles. The costs of publication of this article were defrayed in part by the payment of page Plasmid Constructs. RNAfromthe cell line DH978was usedto prepare charges. This article must therefore be hereby marked advertisement in accordance with p21 eDNA. Both the mutant and the wild-type alleles were cloned in a TA 18 U.S.C. Section 1734 solely to indicate this fact. cloning vector (Invitrogen). HindIII and NotI were used to release the inserts, I To whom requests for reprints should be addressed, at Lymphoma Biology Section, Bldg. 10/13C2O5, National Cancer Institute, NIH, Bethesda, MD 20892. which were then ligated into the pCEP4 expression vector (Invitrogen). 2 The abbreviations used are: BL, Burkitt's lymphoma; SSCP, single-strand confor RNA Isolation and Northern Analysis. Exponentially growing cells (1â€” mational polymorphism; RT-PCR, reverse transcription PCR; bp, base pairs(s). 2 x l0@ cells/ml) were divided into 2 aliquots each. One of the aliquots was 1431

WAF//CIPI GENE IN BL

lysed in an SDS buffer, and the protein lysate was used to determine p53 and downstream primers was based upon the observation that the second p21 levels by Western blotting (see below). The other aliquot was used to exon of the mouse p21 gene and exon 2 of the human gene are flanked prepare total RNA by the method described by Chomczynski and Sacchi (13). by introns at similar positions. Therefore, to design appropriate prim Briefly, cells were lysed in guanidinium thiocyanate, and proteins were cx ers for the amplification of the second exon we initially used, in tracted in the organic phase using acidic phenol. RNA was recovered from the conjunction with the 5' primer (waf-1), 3' primer homologous to the aqueous phase by precipitation with isopropanol and quantitated using UV spectrophotometry at A = 260 nm. Equal amounts of the RNA were electra region encompassed by codons 125 to 131 (waf-131), which we phoresed in a 1.2% agarose/2.2 M formaldehyde gel, transferred to a nylon anticipated from the mouse data to be close to the 3 â€˜endof the exon. membrane, and hybridized with a 32P-labeled p21 cDNA. The cDNA for p21 We then amplified the third exon coding regions by using a 5' primer was obtained by an RT-PCR reaction, cloned in a TA cloning vector, and homologous to codons 135â€”140(waf-140) and a 3' primer homolo sequenced to confirm the identity of the insert. gous to a region 32 bp downstream of the termination codon (waf Western Blotting. To measure p53 and p21 protein, cells in log phase RT-3) in the untranslated region of the p21 mRNA. Expected ampli growth were collected by centrifugation, washed once with PBS, and then fication fragments of 420 bp were obtained in a PCR reaction using lysed on ice for 30 mm in 1% NP4O prepared in PBS that contained 10 g.tg/ml either of the 5' primers and waf-131. The primers for the third exon aprotinin, 10 @Wmlleupeptin,2 mM 4-(2-aminoethyl)benzenesulfonyl fluo ride, 1 mM sodium o-vanadate, 10 mM sodium fluoride, and 5 m@isodium PP1. (waS 140 and waf RT-3), however, amplified a band that was approx The samples were centrifuged at 4Â°C,and the supernatants were transferred to imately 900 bp longer than the expected 135-bp fragment, suggesting new tubes, frozen on dry ice, and stored at â€”80Â°C.Toperform Western blots, the presence of an intron within this fragment. Sequence analysis of thawed cell lysates were boiled for 5 mm in SDS-loading buffer. Total cell this fragment revealed the 3' exon/intron boundary of the second exon protein (100 @.tg)fromeach sample was loaded onto 15% SDS-polyacrylamide is at codon 148 in the human gene. Having determined the extent of gels. Following electrophoresis, the proteins were transferred electrophoreti exon 2 of the p21 gene, we designed primers to analyze the entire cally to an Immobilon membrane (Millipore, Bedford, MA). Membranes were coding region by SSCP. SSCP analysis was performed in four sepa then blocked for 30 mm in 5% skimmed milk at room temperature. A rate sets: one that covered the immediate 5' region and the first SO monoclonal mouse antibody to p53 (jAbl8Ol; Oncogene Science, Uniondale, amino acids (region 2.1); a second fragment covering amino acids 21 NY) was used for p53 protein determination. For p21 protein determination, the membrane was probed with a polyclonal rabbit anti-human Sdil (p21) to amino acids 131 (region 2.2); a third fragment that overlapped with antibody (PharMingen, San Diego, CA). Antibody binding was detected region 2.2 and covered amino acids 111 to 148; and region 3.1 that with chemiluminescence detection procedures according to the manufacturer's encompassed exon 3. Several BL DNA samples showed an abnormal recommendations (Amersham, Amersham, UK). migration of PCR-generated SSCP for region 2.1. The abnormally migrating band was identical in these samples and was frequently Results and Discussion detected in BL DNA samples from Africa. Several DNA samples, SsCPAnalysisofthep21CodingRegionIndicatesThatp2! Is obtained from the peripheral blood lymphocytes of healthy volunteers Most Frequently Wild Type in BL. The p21 gene consists of three from the African continent, also demonstrated this mutation. Only one exons of 68, 450, and 1600 bp, respectively (4). The translation sample, DH978, demonstrated abnormal migration for region 2.2 (Fig. initiation signal is in the second exon, which is also the longest coding IA). Both normal and aberrant conformers were present, indicating exon. In the absence of data pertaining to the complete genomic that the mutation was heterozygous. No abnormal migration was seen organization of the p21 gene on chromosome 6p2l.2 in humans, we for the other regions. relied upon a PCR strategy to determine the most 3' distal sequences Sequencing of Aberrant Conformers. Mutations suggested by in exon 2 that we could use to amplify portions of the human p21 the abnormal migration of coding region fragments from the 2.1 gene. A primer that was homologous to the region 12 bases upstream region were further assessed by sequencing several representative of the initiation codon (waf-1) was synthesized. The design of the DNA samples with aberrant migration (Fig. 1A). An amino acid

A @1JJI @ @_â€”@ @&- @- Fig. 1. A, SSCP analysis of DNA from tumor biopsies of BL. Left, SSCP analysis for the 2.1 w@ â€”@ amplified fragments. Identical migration of the ab . .,,..@---â€”---w- errant band is evident in BL samples 3751, AG876, and BJAB. In the right panel, which is represent ative pattem for the SSCP analysis of fragment 2.2, only DH978 shows abnormal migration. Wt, wild

type DNA. B, sequencing of mutant products. Di â€”@ rect sequencing of the PCR products obtained from amplification of the genomic DNA of DH978 and a control cell line is shown in the left panel. The middle panel shows the sequencing of representa B tive subcloned PCR products obtained from ampli @ DH978 WI DH978 &@ WI AG876 fication of the DH978 mRNA. The right panel C.) C) â€˜..l:, ----@- @, @â€˜ @ shows direct sequencing of the PCR products, with CD AGCTJAGCT 8 AGCT!AGCT c.@ AGCTIIAGCT @@@variant and invariant SSCP patterns for the 2.1 ;@I@ @â€”-J/@Â§@,,.jâ€˜ region for AG876 and a control DNA.

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change from Ser to Arg at codon 31 resulting from a C-A mutation p21BLTable I Growth suppression mediated by wild-type and codon 63 mutated was detected in all the samples that carried additional conformers in ofeithercell lines Ca46 and PA682-PE were transfected by electroporation using 10 @g vector DNA (pCEP4)@ vector DNA containing wild-type p21 (p21), or vector DNA the SSCP analysis. This change was seen most frequently in all BL containingandselection mutant p21 (p21 3)â€¢Colonieswere counted 10 days following transfection samples originating from Africa. Two BL cell lines from North ofthe in hygromycin. The number of colonies shown is an average for two isolates America (EW36 and JLP1 19) also carried this mutation, as did several cell lines obtained from a total of 24 wells plated followingtransfection.Colonies each DNA samples obtained from normal healthy volunteers, suggesting obtainedCell that it is a polymorphism. This mutation results in the gain of a p2163PA682-PEline EBVâ€• p53 pCEP4 p2! restriction site for the enzyme Esp3I. Since this polymorphism was + mut21204 186 4 present in almost all the endemic BL samples, we decided to deter 32CA46 7 mine its frequency in normal DNA samples available from Africa and â€” mut 23522167 6 North America. Our results suggest a frequency of 0.5 in DNA 23a 7 obtained from Nigerians, and 0.3 in DNA from Egyptians. None of the EBV, Epstein-Barr virus. 10 samples obtained from North America showed the polymorphism. We also sequenced the aberrantly migrating 2.2 fragment from DH978 DNA and detected a heterozygous missense mutation (TTC presence in PCR-amplified fragments from the genomic DNA (Fig. TTA) at amino acid 63, which changes Phe to Leu. Codon 63 lies in 2). We next wished to determine whether both alleles (wild-type and the overlapping region in fragments 2.1 and 2.2, but interestingly, the mutant) were expressed. RT-PCR reactions were carried out using SSCP analysis of the 2.1 region did not resolve the aberrantcon total RNA preparations from DH978. The PCR products were cloned former, suggesting that mutations in the extreme ends of this fragment in a TA cloning vector, and transformants were analyzed. Since the may have been missed by SSCP analysis. Therefore, we repeated the mutation creates a Dde! restriction site, we analyzed 12 transformants SSCP analysis of all tumorsamples thatshowed normalmigrationof by restriction enzyme digestion of the 2.2 amplified fragments. Fifty the 2.1 fragment using two different sets of conditions and different eight % of the transformants carried a mutant allele. primers. The 2.1 fragment was run on both MDE (Hydrolink, AT Effect of Mutation at Codon 63 on Growth-suppressive Prop Biochem, Malvern, PA) and 10% glycerol gels. In addition, an am erties ofp2l. We next determined whether the mutation at codon 63 plified fragment that incorporated both the 2.1 and the 2.2 region was from DH978 cells in the p21 gene affected the ability of the protein to assessed by SSCP analysis. However, the mutation in DH978 was not cause growth suppression. Several BL cell lines were tested for their revealed by any of these manipulations. Therefore, to confirm the ability to proliferate after transfection and overexpression of wild-type wild-type status of the p21 2.1 region of tumors other than DH978, we p21. Full-length p21 wild-type cDNA was isolated and cloned into the also sequenced 12 of the normal conformers. No additional mutations pCEP4 expression vector, under the transcriptional control of a CMV were detected. promoter. BL cell lines were transfected with either wild-type p21, Heterozygous Expression ofp2l in DH978. Since the mutation in wild-type in the antisense orientation, or vector alone. Following codon 63 creates an additional DdeI site, we were able to confirm its transfection, cells were grown in the presence of hygromycin to select transfectants and the number of surviving colonies analyzed after 2 weeks. p21 appeared to cause little suppression of clonogenicity in BL cell lines Namalwa, P3HRI, and BL3O. In contrast, there was marked growth suppression in cell lines CA46, PA682PE, and PA682PB. We used PA682 and CA46 in further experiments designed to determine â€” Â°.â€” ,,â€” the relative degree of suppression caused by overexpression of the C co co mutant protein. Mutant cDNA was isolated from DH978 and cloned 00)0) as described for the wild-type into the pCEP4 expression vector. The â€”II cell lines PA682PE and CA46 were transfected with p21 that was either wild type, mutant, or wild type in the antisense orientation, or @@@ 1000bp transfected with vector alone. Following transfection, cells were @ 7OObp@@ grown in the presence of hygromycin, and the number of colonies were scored. Results of a typical transfection experiment are shown in @ 5OObp@5,@ @. Table 1. The codon 63 mutant p21 showed three to S-fold more clones 400bp Ã©,â€” 280 when compared to wild-type p21, presumably a consequence of the 300bp â€˜r â€” 180 mutation at codon 63. The mutation, however, has not completely lost 200bpâ€•,r â€”100 its suppressive phenotype, since the number of clones obtained with the mutant were eight to nine times less than those obtained with lOObp / @ Iâ€” . vector alone. PA682 clones transfected with mutant p21, wild type, or 5Obp vector alone were assayed by Northern and Western Blot analysis to confirm over expression of p21. As seen in Fig. 3A, pooled clones from both mutant and wild-type transfectants demonstrated increased Fig. 2. Restriction analysis of the 2.2 amplified fragment from the BL sample DH978. levels of p21 transcript compared to levels of RNA in pooled trans Total RNA from DH978 was used to prepare cDNA as described in â€œMaterialsand fectants of vector alone. Like the parent PA682PE (data not shown), Methods.â€• The cDNA was used along with primers waf-1 and waf-131 to amplify a 404-bpfragmentencodingexon2 of the gene.GenomicDNAfromperipheralblood clones transfected with vector alone had little or no detectable mes lymphocytes and from DH978 was also used as a template to obtain the 404-bp fragment. sage. It was of interest that the levels of RNA in the mutant transfec The amplified fragment was then digested with DdeI, and the restricted DNA was run on a 4% agarose gel. Lanes 1 and 5 (from left) contain a 100-bp DNA ladder. Lane 2. the tants was 10â€”20-foldhigher than the levels obtained for wild type. restriction analysis of the amplified fragment from control DNA. Lane 3 (amplification Clearly, if the overexpression of p21 is incompatible with growth of from genomic DNA from DH978) and Lane 4 (amplification from a cDNA product from the PA682PE cell line, selection of clones in hygromycin will be DH978 RNA) both demonstrate an extra 18O-bpfragment resulting from the creation of a DdeI site in the mutant allele and retention of the control pattern, consistent with biased towards cells in which expression is moderate to minimal. heterozygosity of the mutation. Although some clones were obtained from PA682 transfected with 1433

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@@@@ .â€˜ @â€˜i@S. Â°â€˜@;-@@â€˜::.@â€¢ â€˜ @, .;â€¢ ..,.&-. - __@,.e@-,-â€¢@â€˜@ @ .@ .Ã .@ . 4 I â€œ p21 â€”@ 4. at, @v ,@ â€¢

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Fig. 3. A, total RNA was extracted from pools of clones of PA682PE, transfected with either the pCEP4 vector alone (Lane C) or with the wild-type cDNA in pCEP4 (Lane Wt) or the mutant cDNA in pCEP4 (Mut). Twenty @gof RNA were electrophoresed in a denaturing agarose gel and transferred to nylon membrane. The blot was hybridized with radiolabeled full-length cDNA probe; panel b depicts the autoradiogram following hybridization. An ethidium bromide staining (a) of the RNA gel is shown to depict equal loading of RNA samples. In addition, (c) depicts protein levels of several transfectedclones as compared to the parent cell line. B, photomicrographsof various clones from transfected PA682PE cell line in culture 3 days after plating at a density of 5 X 10-icells/mi in a 24-well plate. a, representation of cells transfected with vector alone; b, cells transfected with full-length wild-type p21 in the antisense orientation in pCEP4; c, cells transfected with wild-type cDNA in the sense orientation; d, cells transfected with the mutant in the pCEP4 vector.

wild-type p2l, these clones grew poorly, even after subculture, cam Our findings raise the important question as to why p53 mutations, pared to the clones transfected with the mutant p21 or vector control. but not p21 mutations, are often seen in BL. It is possible that the Immunohistochemistry was used to determine if there was any dii contribution ofp53 mutations to BL pathogenesis derives additionally ference in the localization ofp2l in the clones transfected with either from inhibition of alternate p53-controlled pathways unrelated to p21. the mutant or the wild-type construct. Sdil monoclonal antibody It is known, for example, that p53 controls the ability of a cell to (PharMingen) was used in a dilution of 1:50 with antigen retrieval undergo apoptosis by inducing Bax and thus altering the Bcl2:Bax using microwave treatment. In both the sets of clones, the pattern of ratio (14, 15). Based on present knowledge, mutations in p53 would staining was predominantly in the nucleus, with rare cells showing result not only in the loss of DNA damage-induced G1 arrest but also cytoplasmic staining (data not shown). Thus, the mutant p21 does not in the loss of DNA damage-induced apoptosis, irrespective of whether appear to compartmentalize in the cytoplasm. the two pathways are separated or not. If the apoptosis pathway does When equal numbers of cells from the transfected clones were not involve p21, mutations in p21 would only result in loss of G@ plated, we observed that close to 50% of cells took up trypan blue in arrest, but no inhibition of DNA damage-induced apoptosis (in the wild-type transfectants by day 3 following subcloning, whereas cells presence of wild-type p53). The retention of an intact apoptotic transfected with either the mutant or vector alone showed little if any pathway triggered by DNA damage may limit the level of genomic trypan blue uptake. The suppressive properties of the wild-type p21 instability and hence the rate of tumor progression, perhaps explaining cannot be attributed to nonspecific toxicity of the plasmid prepara tions, because the same preparations did not demonstrate any growth the lack of p21 mutations in BL. Alternatively, p21 might be an suppression in at least three other BL cell lines. Morphological as essential gene whose loss could limit survival; the phenotype of p21 sessment of the cells transfected with wild-type p21, however, clearly knockout mice would help to elucidate this further. We have shown demonstrated increased numbers of cells manifesting a granular ap previously that BL cell lines that contain mutated p53 fail to G1 arrest pearance. Clones transfected with either the mutant or the vector or undergo apoptosis following DNA damage following irradiation or alone, in contrast, appeared healthy (Fig. 3B). DNA fragmentation cytotoxic drug exposure (16, 17). We believe that this enables BL experiments used to assess the fraction of apoptotic cells in the three cells to resist chemotherapy-induced apoptosis, a hypothesis which is transfectants failed to reveal an increase in apoptotic cells in the supported by the finding (3) that the presence of p53 mutation is a clones carrying wild-type p21. Thus, the poor viability of wild-type poor prognostic factor in BL and other tumors. The lack of mutations p21 transfectants cannot be directly attributed to death by apoptosis as in p21 in BL, which should also cause lack of G@arrest following assessed by DNA fragmentation (data not shown). DNA damage, suggests that abrogation of the p53-dependent apoptic 1434

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. WAFI/CIPI GENE IN BL pathway, rather than the loss of G@arrest and DNA repair is the reason 9. Gu, Y., Turck, C., and Morgan, D. Inhibition of Cdk2 activity in vivo by an associated for chemo- and radioresistance in BLs carrying mutated p53 gene. 20K regulatory subunit. Nature (Land.), 366: 707â€”710,1993. 10. Waga, S., Hannon, 0., Beach, D., and Stillman. B. The p21 inhibitor of cyclin dependent k.inases controls DNA replication by interaction with PCNA. Nature References (Land.), 369: 574-578, 1994. 11. Gutierrez, M., Bhatia, K., Barriga, F., Die; B., Sackman Muriel, F., Andreas, M., 1. Gaidano, G., Ballerini, P., Gong, 3., lnghirami, G., Neri, A., Newcomb, E., Magrath, I., Knowles,D.,andDalla-Favera,R.p53mutationsinhumanlymphoidmalignan Epelman, S., Risueno, C., and Magrath, I. Molecular epidemiology of Burkitt's cies:associationwithBurkitt'slymphomaandchroniclymphocyticleukemia.Proc. lymphoma from South America: differences in breakpoint location and Epstein-Barr Nati.Acad.Sd. USA,88: 5413â€”5417,1991. virus association from tumors in other world regions. Blood, 79: 3261â€”3266,1992. 12. Bhatia, K., Gutierrez, M., Huppi, K., and Magrath, I. PCR detection of a neutral 2. Bhatia, K., Gutierrez, M., Huppi, K., Siwarski, D., and Magrath, I. The pattern of p53 mutations in Burkitt's lymphoma differs from that of solid tumors. Cancer Res., 52: CGA/CGG dimorphism in exon 6 of the human p53 gene. Nucleic Acids Res., 20: 4273â€”4276,1992. 928, 1992. 3. Gutierrez, M., Bhatia, K., Diez, B., Sackman Muriel, F., Epelman, S., Dc Andreas, 13. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid M. L, Huppi, K., and Magrath, I. Prognostic significance of p53 mutations in small guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: non-cleaved cell lymphomas. tat. J. Oncol., 4: 567â€”571,1994. 156â€”159,1987. 4. Ei-Diery, w., Tokino, T., Veiculescu, V., Levy, D., Parsons, R., Trent, J., Un, D., 14. Miyashita, T., Krajewski, S., Krajewski, M., Wang, H., Lin, H., Lieberman, D., Mercer, E., Kinzler, K., and Vogeistein, B. WAF1 a potential mediator of p53 tumor Hoffman, B., and Reed, J. Tumor suppressor p53 is a regulator ofbcl-2 and box gene suppression. Cell, 75: 817â€”825,1993. expression in vitro and in vivo. Oncogene, 9: 1799â€”1805,1994. 5. Noda, A., Ning, Y., Venable, S. F., Percira-Smith, 0. M., and Smith, J. R. Cloning of 15. Sevakumaran, M., Un, H., Miyatashita, T., Wang, H., Krajewski, S., Reed, J., senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Hoffman, B., and Lieberman, D. Immediate early up-regulation of bas expression by Cell Res., 11: 90â€”98,1994. p53 but not TGFB1: a paradigm for distinct apoptotic pathways. Oncogene, 9: 6. Harper, J. W. Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. The p2! 1791â€”1798,1994. Cdk-interacting protein Cip! is a potent inhibitor of Gi cyclin-dependent kinases. 16. O'Connor, P., Jackman,J., Jondle,D., Shatia, K., Magrath, I., and Kahn, K. Role of Cell, 75: 805â€”816,1993. p53 tumor suppressorgenein cell cycle arrestand radiosensitivityofBurkitt's 7. Xiong, Y., Hannon, G. J., Zhang, H., Casso D, Kobayashi, R., and Beach D. p2! is lymphomacelllines.CancerRca.,53: 4776â€”4780,1993 a universal inhibitor of cyclin kinases. Nature (Land.), 366: 701â€”704,1993. 17. Fan, S., El-Deiry, W., Freeman, I., Jondle, J., Bhatia, K., Magrath, I., Fornace, A. J., 8. Serrano, M., Hannon, G., and Beach, D. A new regulatory motif in cell cycle Jr., Kohn, K., and O'Connor, P. p53 gene mutations are associated with decreased control causing specific inhibition of cyclin D/CDK4. Nature (Land.), 366: sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res., 54: 704â€”707,1993. 5824â€”5830,1994.

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Kishor Bhatia, Saijun Fan, Gordon Spangler, et al.

Cancer Res 1995;55:1431-1435.

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