P53 Induction Prevents Accumulation of Aberrant Transcripts in Cancer Cells1

[CANCER RESEARCH 61, 486–488, January 15, 2001] Advances in Brief p53 Induction Prevents Accumulation of Aberrant Transcripts in Cancer Cells1

Caroline Moyret-Lalle, Cyril Duriez, Joris Van Kerckhove, Christel Gilbert, Qing Wang, and Alain Puisieux2 De´partement d’Oncologie Fondamentale et Applique´e, INSERM Unite´453, Centre Le´on Be´rard, 69008 Lyon, France

Abstract Materials and Methods

Loss of fidelity of the splicing process occurs during tumor progression Cell Lines and Treatments. Parental cell lines used in these studies were and can have a deleterious effect on genes like tumor suppressor genes. It obtained from the American Culture Collection (Rockville, MD). Breast was reported recently that the presence of aberrant transcripts of the HBL100 and colon HCT116 cancer cells were grown in McCoy’s medium TSG101 gene in breast cancer cells was associated with the mutation of the (Life Technologies, Inc.) supplemented with 10% FCS. Breast adenocarci- p53 tumor suppressor gene. On the basis of this observation, we have noma MCF7 and MDA-MB-231 cells and colon carcinoma EB cells were analyzed TSG101 transcript patterns in p53-active and p53-inactive cells. maintained in DMEM (Life Technologies, Inc.) containing 10% FCS. Hepa- Using several isogenic cellular models, we demonstrate that the induction tocarcinoma HepG2 and Hep3B cells were maintained in Eagle’s MEM (Life of p53 in cancer cells leads to a significant decrease of aberrant transcripts Technologies, Inc.) containing 10% FCS. Esophagus cancer TE-1 cells were cultured in RPMI 1640 supplemented with 10% FCS. Parental MCF7, levels. This indicates a novel implication of p53 in the regulation of the HBL100, HepG2, and HCT116 cell lines express an endogenous wild-type p53 splicing process. gene. MDA-MB-231 and TE-1 cells express a mutant p53, whereas Hep3B exhibits an homozygous deletion of the gene. Cells were plated at 106 cells/ Introduction 100-mm dish 24 h before radiation treatment. Cells were exposed to ␥-irradiation at 6 Gy (a high energy X-irradiation, 5 or 10 MV, at ϳ3 Gy/min). Tumor development is driven by genetic and epigenetic events RNA Isolation and RT-PCR.3 Total RNA was extracted using Tri- leading to the alterations of oncogenes and tumor suppressor genes. Reagent (Sigma Chemical Co.). cDNA was synthesized from 3 ␮g of total During the last few years, several reports have demonstrated that RNA with first-strand cDNA synthesis kit (Amersham Pharmacia Biotech). carcinogenesis induced a deregulation of the process of alternative The reaction mixture was incubated at 37°C for 1 h, and cDNA was stored at splicing (1). Tumor suppressor genes can be affected by such abnor- Ϫ70°C. Nested RT-PCR reactions were carried out using the conditions malities, suggesting that splicing defects may participate in the ma- described by Li et al. (2). P1 (5Ј-CGGGTGTCGGAGAGCCAGCTCAA- Ј Ј Ј lignant transformation. The TSG101 gene was first identified as a GAAA-3 ) and P2 (5 -CCTCCAGCTGGTATCAGAGAAGTCGT-3 ) primers were used for the first PCR round for 30 cycles. Second-round PCR for 25 candidate tumor suppressor gene, based on the observation of intra- cycles was performed using two nested primers, P3 (5Ј-AGCCAGCTCAA- genic deletions in breast cancers (2). These results were denied by GAAAATGGTGTCCAAG-3Ј) and P4 (5Ј-TCACTGAGACCGGCAGTCTT- later studies that failed to demonstrate any genomic rearrangements of TCTTGCTT-3Ј). The RT-PCR products were analyzed in 1.5% agarose gel. the gene (1, 3). However, it is now clear that TSG101 is a frequent cDNA Subcloning and Sequencing. Aberrant PCR products were cut out target of splicing defects. Although aberrant transcripts may be found and purified with the Concert Rapid gel extraction system (Life Technologies, in normal tissues, the frequency of TSG101 splicing abnormalities Inc.). Purified cDNAs were cloned in the pGEM-T vector (Promega Corp.) and increases during transformation and are consistently observed in sequenced using an ABI PRISM 377 DNA sequencer (Perkin-Elmer). Se- quencing was carried out in both directions with the P3 and P4 primers. breast, cervix, prostate, liver, and gastrointestinal cancers compared with adjacent normal tissues (2, 4–6). Variant transcripts found in Results and Discussion tumors are generally shorter than the wild-type mRNA. They are generated by exon skipping and alternate RNA processing events. Presence of the ⌬154–1054 TSG101 Splice Variant in Cancer Increasing levels of variant transcripts in tumors could reflect a Cells. To study the presence of TSG101 splice products in relation progressive loss of stringent splice control function or an extended with p53 status, we first examined the expression TSG101 transcripts alternative splicing during tumor progression. Several studies have by nested RT-PCR as described by Li et al. (2) in wild-type and reported a correlation between the presence of aberrant transcripts and mutant p53 cancer cell lines derived from breast (MCF7, HBL100, tumor grades (1, 4, 5, 7). Recently, Turpin et al. (8) have observed, in and MDA-MB-231), colon (HCT116), and liver (HepG2 and Hep3B) breast cancers, an association of TSG101 aberrant spliced products to tumors. Using primers flanking the TSG101 coding region, the pre- p53 mutation. Overall, these results suggest that an increasing relax- dicted 1192-bp full-length transcript (Fig. 1) was observed in all cell ation of TSG101 splicing fidelity occurs during malignant progres- lines after the first round of PCR amplification. After the second round of PCR, in addition to the 1145-bp, normal-sized TSG101 sion. To address the specific question of whether p53 function can transcript, bands of smaller size were detected in all cell lines (data not interfere with an alternative splicing process, we have analyzed the shown). An ϳ250-bp transcript was observed in most cases as a major presence of aberrant TSG101 transcripts in different isogenic cell aberrant RT-PCR product (Figs. 1 and 2). Southern blot analysis of models differing only in p53 status. the RT-PCR products, using full-length TSG101 cDNA as a probe, confirmed the authenticity of this variant TSG101 splice product (data Received 8/7/00; accepted 12/6/00. not shown). Sequence analysis revealed that this fragment (244 bp) The costs of publication of this article were defrayed in part by the payment of page was the most frequent variant TSG101 form identified previously in charges. This article must therefore be hereby marked advertisement in accordance with tumors (1, 9, 10). It corresponds to a deletion of 900 nucleotides 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Comite´De´partemental de la Ligue de Lutte contre le (nucleotides 154-1054). The deletion junction contains a donor site- Cancer, the Association pour la Recherche sur le Cancer, and INSERM (U453). like sequence within the coding exon 1 and an acceptor site-like 2 To whom requests for reprints should be addressed, at INSERM Unite´453, Centre Leon Berard, 28 rue Laennec, 69008 Lyon, France. Fax: 33-4-78-78-27-20; E-mail: [email protected]. 3 The abbreviation used is: RT-PCR, reverse transcription-PCR. 486

adenocarcinoma MCF7 cells with a dominant-negative p53 minipro- tein (12, 13). This COOH-terminal segment of p53 forms stable oligomers with endogenous p53, leading to the abrogation of sequence-specific DNA binding. MCF7/MN1 cells were generated from MCF7 cells by transfection of the antibiotic resistance gene alone. Endogenous wtp53 was inactivated in HCT116 colon cancer cells by homologous recombination (14). As it was shown in parental cell lines, exposure to X-rays led to a decrease of the ⌬154–1054 truncated transcript levels in p53-active cell lines [MN1 and HCT116 p53(ϩ/ϩ)], whereas levels of the full-length transcript remained unchanged. In contrast, levels of the abnormal transcript were un- affected in the two p53-inactive cell lines [MDD2 and HCT116 p53(Ϫ/Ϫ); Fig. 3]. Induction of p53 Alone Is Not Sufficient to Down-Regulate the ⌬154–1054 TSG101 Truncated Transcript. To further study the regulatory role of p53, we tested the pattern of TSG101 transcripts in three cellular models displaying an inducible p53 activity: Fig. 1. Schematic representation of the ⌬154–1054 deleted TSG101 transcript. The (a) An expression plasmid encoding a temperature-sensitive (ts) coding exons of the normal TSG101 transcript, indicated as E1–E6, as defined by Li et al. version of mutant p53 (murine p53 Val135) under the control of a (2), are shown in gray. The locations of primers P1, P2, P3, and P4 used for nested constitutive promoter was stably transfected into Hep3B, a human RT-PCR amplification of TSG101 are indicated. Deletional breakpoints are indicated by nucleotide numbers. Boldface highlights donor site-like and acceptor site-like sequences. hepatocarcinoma cell line that contains no endogenous p53 protein. The nucleotide numbers are shown according to the nucleotide sequence of TSG101 As described previously, ts-p53 protein is inactive at 39°C but is cDNA, GenBank accession number U 82130. transcriptionally active upon shift to the permissive temperature of 32°C (15). (b) The esophageal cancer cell line TE-1 expresses an endogenous

Fig. 2. RT-PCR analysis of TSG101 transcripts in response to DNA damage. cDNA amplification of full-length (1145-bp) and ⌬154–1054 truncated (244-bp) transcripts by RT-PCR in three breast cancer cell lines (MCF7 and HBL100 express an endogenous wtp53, whereas MDA-MB-231 cells express a mutant p53). Untreated (C) or irradiated (X; 6 Gy) MCF7, HBL100, and MDA-MB-231 cells were tested 24 h after irradiation. (Results were highly reproducible from different irradiation experiments, and all RT-PCR amplifications were performed in triplicate.)

sequence within the coding exon 5 (Fig. 1). This deletion encom- passes the segment encoding the coiled-coil domain of the TSG101 protein. The resulting aberrant transcript alters the open reading frame and introduces several in-frame termination codons. Fig. 3. Kinetic analysis of TSG101 transcripts after X-rays in two cellular models Levels of TSG101 Spliced Variants Decrease in Stress-induced exhibiting experimental inactivation of p53. cDNA amplification of full-length (1145-bp) and ⌬154–1054 truncated (244-bp) transcripts by RT-PCR in MN1/MCF7 and MDD2/ p53 Cells. Wild-type p53 (wtp53) is present at extremely low levels MCF7 cells for 18–48 h after X-rays (A) and in HCT116 p53 (ϩ/ϩ) and p53(Ϫ/Ϫ) cells in most cells. In response to DNA damage, the half-life of p53 is for 0–24 h after X-rays (B). increased, and the protein accumulates in the nucleus where it can act as a transcriptional factor (11). To determine whether activation of p53 function could be involved in regulation of TSG101 splicing, TSG101 product patterns were studied after X-irradiation, using nested RT-PCR in three breast cancer cell lines (MCF7, HBL100, and MDA-MB-231). As expected, the aberrant TSG101 product ⌬154– 1054 was amplified alongside the normal transcript in nonirradiated cells (Fig. 2). Twenty-four h after exposure to X-rays, levels of the truncated transcript significantly decreased in wtp53 cell lines, whereas it remained high in the mutant p53 cell line (Fig. 2). This observation suggested a p53-dependent mechanism. To assess the specific role of p53, we performed a detailed kinetic analysis of Fig. 4. Kinetic analysis of TSG101 transcripts in a p53-inducible cellular model. cDNA TSG101 transcripts in response to ␥-irradiation in MCF7 and amplification of full-length (1145-bp) and ⌬154–1054 truncated (244-bp) transcripts by RT-PCR in Hep3B-tsp53 cells after shifting to the p53-permissive temperature (32°C) for HCT116-derived cell lines, exhibiting an inactivation of endogenous 2–24 h is shown. Cells grown at 39°C (nonpermissive temperature) for 2–24 h were used p53. MCF7/MDD2 cells were generated by stably transfecting breast as controls. 487

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2001 American Association for Cancer Research. p53 AND ABERRANT TRANSCRIPTS mutant p53 (amino acid 272, methionine to valine) that functions as a 3. Wang, Q., Driouch, K., Courtois, S., Champe`me, M-H., Bie`che, I., Treilleux, I., ts-p53 (16, 17). Briffod, M., Rimokh, R., Magaud, J-P., Curmi, P., Lidereau, R., and Puisieux, A. Low frequency of TSG101/CC2 gene alterations in invasive human breast cancers. Onco- (c) The third cellular model, EB1, was derived from the human gene, 16: 677–679, 1998. colon carcinoma cell line EB after stable transfection with a construct 4. Sun, Z., Pan, J., Bubley, G., and Balk, S. P. Frequent abnormalities of TSG101 containing the wild-type p53 cDNA under the control of the metal- transcripts in human prostate cancer. Oncogene, 15: 3121–3125, 1997. 5. Klaes, R., Kloor, M., Willeke, F., Melsheimer, P., Von Knebel Doeberitz, M., and lothionein MT-1 promoter (18). Ridder, R. Significant increase of a specific variant TSG101 transcript during the In all models, p53 induction leads to a cell cycle arrest (data not progression of cervical neoplasia. Eur. J. Cancer, 35: 733–737, 1999. shown). The ⌬154–1054 truncated transcript was detected in addition 6. Chen, Y-J., Chen, P-H., Lin, S-Y., and Chang, J-G. Analysis of aberrant transcription of TSG101 in hepatocellular carcinomas. Eur. J. Cancer, 35: 302–308, 1999. to the normal transcript in the three noninduced cell lines. Levels of 7. Lin, P-M., Liu, T-C., Chang, J-G., Chen, T-P., and Lin, S-F. Aberrant TSG101 truncated transcripts were not modified after p53 induction (Fig. 4), transcripts in acute myeloid leukaemia. Br. J. Haematol., 102: 753–758, 1998. suggesting that wtp53 expression is necessary but not sufficient to 8. Turpin, E., Dalle, B., de Roquancourt, A., Plassa, L. F., Marty, M., Janin, A., Beuzard, Y., and de The´, H. Stress-induced aberrant splicing of TSG101: association to high modulate expression of TSG101 aberrant transcripts. tumor grade and p53 status in breast cancers. Oncogene, 18: 7834–7837, 1999. Our results indicate that stress-activated p53 is able to modulate the 9. Wang, N. M., and Chang, J-G. Are aberrant transcripts of FHIT, TSG101, and TSG101 splicing process. The underlying mechanism remains to be PTEN/MMAC1 oncogenesis related? Int. J. Mol. Med., 3: 491–495, 1999. 10. Oh, Y., Proctor, M. L., Hong Fan, Y., Su, L-K., Ki Hong, W., Fong, K. M., Sekido, elucidated. The lack of change in TSG101 transcripts pattern after Y. S., Gazdar, A. F., Minna, J. D., and Mao, L. TSG101 is not mutated in lung cancer experimental induction of p53 (ts-p53; inducible expression) leads to but a shortened transcript is frequently expressed in small cell lung cancer. Oncogene, two main remarks: (a) this suggests that p53 effects depend either on 17: 1141–1148, 1998. 11. Kastan, M. B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R. W. specific posttranslational modifications induced by genotoxic treat- Participation of p53 protein in the cellular response to DNA damage. Cancer Res., 51: ments or on stress-induced coactivators; and (b) this indicates that the 6304–6311, 1991. variation of the TSG101 spliced transcript levels is not a secondary 12. Shaulian, E., Zauberman, A., Ginsberg, D., and Oren, M. Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence- consequence of the p53-dependent cell cycle arrest. Consistently, specific DNA binding. Mol. Cell. Biol., 12: 5581–5592, 1992. ␥-irradiation triggers a similar decrease of aberrant transcript levels in 13. Bacus, S. S., Yarden, Y., Oren, M., Chin, D. M., Lyass, L., Zelnick, C. R., Kazarov, p21Waf1-proficient and -deficient HCT116 cells (data not shown). A., Toyofuku, W., Gray-Bablin, J., Beerli, R. R., Hynes, N. E., Nikiforov, M., Haffner, R., Gudkov, A., and Keyomarsi, K. Neu differentiation factor (Heregulin) Thus, the activation of this cyclin kinase inhibitor, which is a major activates a p53-dependent pathway in cancer cells. Oncogene, 12: 2535–2547, 1996. p53 target gene, is not required for the p53-dependent modulation of 14. Bunz, F., Dutriaux, C., Lengauer, C., Waldman, T., Zhou, S., Brown, J. P., Sedivy, splicing process. J. M., Kinzler, K. W., and Vogelstein, B. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science (Washington DC), 282: 1497–1501, 1998. 15. Rouault, J-P., Falette, N., Guehenneux, F., Guillot, C., Rimokh, R., Wang, Q., Acknowledgments Berthet, C., Moyret-Lalle, C., Savatier, P., Pain, B., Shaw, P., Samarut, J., Magaud, J-P., Ozturk, M., Samarut, C., and Puisieux, A. p53-dependent induction of the We thank M. Oren and K. Keyomarsi for MN1 and MDD2 cells, B. antiproliferative BTG2 gene, a component of the DNA damage cellular response. Nat. Vogelstein for HCT116 p53 (Ϫ/Ϫ) and p21 (Ϫ/Ϫ) cells, and P. Hainaut for Genet., 14: 482–486, 1996. TE-1 cells. We also thank C. Ginestet, F. Lafay, and C. Malet for performing 16. Barnas, C., Martel-Planche, G., Furukawa, Y., Hollstein, M., Montesano, R., and the ionizing radiation. Hainaut, P. Inactivation of the p53 protein in cell lines derived from human esophageal cancers. Int. J. Cancer, 71: 79–87, 1997. References 17. Cortes, U., Moyret-Lalle, C., Falette, N., Duriez, C., El Ghissassi, F., Barnas, C., Morel, A-P., Hainaut, P., Magaud, J-P., and Puisieux A. BTG genes expression in the 1. Lee, M. P., and Feinberg, A. P. Aberrant splicing but not mutations of TSG101 in p53-dependent and the p53-independent cellular response to DNA damage. Mol. human breast cancer. Cancer Res., 57: 3131–3134, 1997. Carcinog., 27: 57–64, 2000. 2. Li, L., Li, X., Francke, U., and Cohen, S. N. The TSG101 tumor susceptibility gene 18. Shaw, P., Bovey, R., Tardy, S., Sahli, R., Sordat, B., and Costa, J. Induction of is located in chromosome 11 band p15 and is mutated in human breast cancer. Cell, apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc. Natl. 88: 143–154, 1997. Acad. Sci. USA, 89: 4495–4499, 1992.

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Caroline Moyret-Lalle, Cyril Duriez, Joris Van Kerckhove, et al.

Cancer Res 2001;61:486-488.

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