CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Righetti, S. C.
Right arrow Articles by Zunino, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Righetti, S. C.
Right arrow Articles by Zunino, F.
Cell Growth & Differentiation Vol. 10, 473-478, July 1999
© 1999 American Association for Cancer Research

Emergence of p53 Mutant Cisplatin-resistant Ovarian Carcinoma Cells following Drug Exposure:Spontaneously Mutant Selection1

Sabina C. Righetti, Paola Perego, Elisabetta Corna, Marco A. Pierotti and Franco Zunino2

Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have previously shown that p53 mutations are associated with cisplatin resistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely elucidated; in particular, the biological mechanisms behind the acquired drug-resistant p53-mutant phenotype were not clearly explained. Thus, in this study, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (270 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexistent in the cisplatin-sensitive parental cell line (IGROV-1) or were induced by drug (genotoxic) treatment. For this purpose, an allele-specific PCR approach was used. Primers carrying the desired mutations (T->A codon 270, C->T codon 282) in the 3' terminus, and the corresponding wild-type primers were used to amplify genomic DNA from the original IGROV-1 cell line used to select the mutant IGROV-1/Pt 1. To increase sensitivity, we hybridized blots of the PCRs with the radiolabeled PCR fragment from IGROV-1/Pt 1. Amplification was obtained for IGROV-1 DNA with the mutated allele-specific primers, indicating the preexistence of a mutated population in the IGROV-1 cell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%. Single-strand conformation polymorphism and allele-specific PCR analysis of the IGROV-1/Pt 0.1 cells, which are less resistant to cisplatin than IGROV-1/Pt 1 cells and which carry both mutant and wild-type p53 alleles with a wild-type predominance, suggested a progressive selection of the mutant population by cisplatin treatment. This is the first observation that indicates that a subpopulation of p53 mutant cells can occasionally be selected by cisplatin treatment. Thus, considering the susceptibility to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, the selection process resulting in emergence of p53 mutant tumors is a possible origin of resistance of ovarian carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cisplatin is among the most effective agents that are clinically available for the treatment of a variety of solid tumors, including ovarian carcinoma. However, the development of drug resistance is a common problem for the efficacy of the pharmacological treatment (1 , 2) . Different mechanisms may contribute to define the resistant phenotype, including alterations in drug-target interactions, expression of defense and/or detoxification mechanisms, and cellular responses to DNA damage (3, 4, 5) . In particular, it has been proposed that reduction of the apoptotic response is a critical determinant of cisplatin efficacy (6 , 7) . Wild-type p53 is an important component of the pathway leading from DNA damage to apoptosis because p53 protein is implicated in multiple functions that include control of cell cycle, DNA repair, cell senescence, genomic stability, and stress responses (8 , 9) . Mutations of the p53 gene are common alterations found in a variety of human tumors (10) . Although loss of normal p53 function can confer resistance to DNA-damaging agents as a consequence of a reduced cell susceptibility to apoptosis, the relevance of p53 mutations in chemosensitivity remains controversial (11, 12, 13) . Several studies indicate that p53 can be inactivated in cisplatin-resistant cell systems (14 , 15) . Clinical studies support a correlation between missense mutations and resistance to platinum drug therapy (16) .

On the basis of these observations, the aim of this study was to clarify whether, in an ovarian carcinoma p53 mutant cisplatin-resistant variant, IGROV-1/Pt 1, the presence of p53 mutations was a consequence of the genotoxic treatment or resulted from a selection process. For our purpose, we used allelic-specific gene amplification by PCR. These results support that the p53 allele carrying a mutation at codon 270 preexisted in the IGROV-1 parental cell line with a frequency <0.1% and that the cell population exhibiting the mutation can occasionally be selected by cisplatin treatment.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cellular Sensitivity to Cisplatin.
Fig. 1Citation shows cellular sensitivity to cisplatin of IGROV-1 cells and cisplatin-resistant sublines, including the p53 mutant IGROV-1/Pt 1 and IGROV-1/Pt 0.1 cells. IGROV-1/Pt 0.1 cells, which represent an early step in the process of selection of the IGROV-1/Pt 1 variant, exhibited a degree of resistance of 4. The IGROV-1/Pt 1 variant, in addition to carrying mutations in the p53 gene at codons 270 and 282, exhibits a reduced expression of Bax (14) and increased expression of Bcl-2 (17) . The molecular features of the resistant sublines is consistent with a reduced susceptibility to cisplatin-induced apoptosis (14) .



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. Dose-response curves for the antiproliferative effects of cisplatin against ovarian carcinoma IGROV-1 cells and cisplatin-resistant sublines. Sensitivity to cisplatin was assessed by tetrazolium dye (MTT) assay after 96 h exposure. IC50, drug concentrations required for 50% inhibition of cell growth. RI, resistance index (ratio of IC50 of parental sensitive cells to IC50 of resistant cells).

 
p53 Gene Analysis of IGROV-1/Pt 0.1 Cells.
A molecular analysis of p53 status of IGROV-1/Pt 0.1 cells by SSCP3 analysis of exon 8 revealed the presence of two bands with altered mobility in comparison to control DNA (Fig. 2)Citation . Such bands corresponded to mutant alleles representing mutations at codons 270 and 282, because they comigrated with the mutant alleles previously detected in the IGROV-1/Pt 1 variant (14) . The frequency of the mutant alleles in IGROV-1/Pt 0.1 cells was lower than in the IGROV-1/Pt 1 subline, as indicated by the weakness of the bands with altered mobility, showing that, in the IGROV-1/Pt 0.1 subline, mutant alleles with a predominance of wild-type alleles were concomitantly present.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 2. p53 gene status in the IGROV-1 cell systems. p53 gene status was evaluated by SSCP analysis. Exon 8 analysis is reported.

 
Allele-specific PCR.
We have shown earlier that the IGROV-1/Pt 1 cell line carries two mutant alleles at exon 8 of the p53 gene, involving codons 270 (T->A) and 282 (C->T; Ref. 14 ). To determine whether a small population of p53 mutant cells existed in the parental IGROV-1 cell line before selection with cisplatin, primers carrying the desired mutations in the 3' terminus and the corresponding wild-type primers were used in allele-specific PCR experiments. As shown in Fig. 3Citation , mutation-specific primers specifically amplified the p53 mutagenized sequences contained in the pC53-M plasmids (Lane 4), thus ruling out the possibility of nonspecific amplification of wild-type sequence. Similarly, amplification was specific also with wild-type p53 sequence primers (Lane 1). When the wild-type primers were used to amplify genomic DNA from the cisplatin-sensitive IGROV-1 cells and the two cisplatin-resistant variants IGROV-1/Pt 0.1 and IGROV-1/Pt 1, amplification of all of the templates was obtained (Fig. 4)Citation . Such primers were expected to amplify also genomic DNA of the resistant sublines because the wild-type allele is still present in these cells (Fig. 2)Citation . With the mutated primers amplification was observed for all of the ovarian carcinoma cell lines (Fig. 4)Citation . However, the intensity of the amplified band was different, the highest being observed for IGROV-1/Pt 1 cells. Amplification was intermediate between IGROV-1 and IGROV-1/Pt 1 cells for the IGROV-1/Pt 0.1 cell variant. Thus, the intensity of the amplified band paralleled the amount of p53 mutation present based on SSCP analysis in cisplatin-resistant cells, suggesting that the mutation existed before drug selection in the IGROV-1 cell line. Titration experiments were performed to estimate the proportion of p53 mutated cells preexistent in the IGROV-1 cell line. Genomic DNA from IGROV-1/Pt 1 was mixed with the DNA of wild-type p53 cells, at different ratios ranging from 1:10 to 1:10,000. As shown in Fig. 5Citation , allele-specific PCR of these templates revealed bands with an intensity corresponding to the amount of the mutated DNA present in the mixture, with a detection limit of 0.001. The intensity of the IGROV-1 band, amplified with both the mutation-specific primers, corresponded to the signal of the band amplified when DNA from mutant and wild-type cells were mixed at a ratio between 1:10 and 1:100 (Fig. 5)Citation . False-positive reactions caused by contamination of DNA or nonspecific amplification of wild-type sequences were ruled out by appropriate controls. Although these results were consistently reproduced, allele-specific PCR revealed no amplification using as template other independent DNA extractions from IGROV-1 cell line. Therefore, to further increase detection sensitivity, we performed blots of allele-specific PCR. At the annealing temperatures used for non-hot PCR, weak positive signals were observed also with DNA from cell line carrying wild-type p53, suggesting that, at these temperatures, the primers were not completely destabilized. As shown in Fig. 6ACitation , with the 270 mutated primer higher annealing temperature (68°C) could eliminate amplification on different wild-type p53 DNA templates. However, under this condition, amplification occurred for four independently prepared IGROV-1 templates (Fig. 6BCitation ; IGROV-1 I–IV), the highest intensity being confirmed for IGROV-1 I. Titration experiments confirmed that the intensity of the IGROV-1 I band corresponded to a ratio between 1:10 and 1:100 of the reference mixtures, whereas for the other three extractions, it was lower than 0.001, indicating that <0.1% of the sample represented preexistent population with p53 mutation. Blot analysis of allelic-specific PCR experiments, performed with the 282 mutated primer, at its own annealing temperature, also revealed weak amplification on wild-type templates, including negative controls, although the amplification was lower than that obtained with the IGROV-1 DNA. However, under this extreme condition, higher annealing temperatures also completely abolished signals on the IGROV-1 templates (data not shown).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3. Allelic-specific PCR on plasmid templates. A, PCR for the 270 mutation (primers 6As/p53-270w or p53-270m); B, PCR for the 282 mutation (primers 6As/p53-282w or p53-282m). Ten ng of plasmid DNA were amplified (30 cycles). Sample loading was as follows: Lane 1, wild-type p53 plasmid + wild-type primers; Lane 2, mutated p53 plasmid + wild-type primers; Lane 3, wild-type p53 plasmid + mutated primers; Lane 4, mutated p53 plasmid + mutated primers: Lane 5, no DNA + wild-type primers; Lane 6, no DNA + mutated primers; Lane M, markers.

 


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Allelic-specific PCR analysis of the p53 270 mutation on genomic DNA. Lane M, markers; Lanes 1, primers p53-8.3/p53-270w; Lanes 2, primers p53-8.3/p53-270m; Lane -, negative control (no DNA).

 


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 5. Allelic-specific PCR analysis: titration experiments. The genomic DNA of IGROV-1/Pt 1 was mixed with DNA from wild-type p53 cells at various ratios. A, primers p53-8.3/p53-270m; B, primers p53-8.3(-7)/p53-282m. Lane M, markers; Lane -, no DNA.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6. Allelic-specific PCR analysis of the p53 270 mutation on genomic DNA: blot with labeled DNA fragment from IGROV-1/Pt 1. A, negative controls (wild-type p53 cells) and IGROV-1 cells. B, genomic DNA of IGROV-1/Pt 1 cells was mixed at different ratio with DNA from wild-type p53 cells (H460).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
p53 is one of the most frequently mutated genes in human tumors (18) , including advanced ovarian carcinoma. An association between p53 mutations and resistance to DNA-damaging agents has been documented in both preclinical and clinical studies (14 , 16 , 19) . Because induction or selection of mutations is a controversial aspect of development of drug resistance, a problem that has remained unsolved since 1979 (20) , in this study, we took advantage of a previously selected cisplatin-resistant cellular system IGROV-1/Pt 1, characterized by mutant p53, to address the issue of whether cisplatin resistance results from a selection of a small fraction of p53 mutant cells preexisting in the original drug-sensitive cell line prior exposure to increasing drug concentrations. For this purpose, sensitive procedures that are able to discriminate between the wild-type and the mutated populations were used. In particular, allelic-specific gene amplification by PCR was chosen because it allows detection of mutations below the detection threshold of PCR-SSCP. Unfortunately, the success of this technique is tightly dependent on the type of mutation, and appropriate conditions cannot be worked out for all of the mutations. These results are consistent with the interpretation that the p53 mutations found in the cisplatin-resistant IGROV-1/Pt 1 cell line preexisted in the parental IGROV-1 cells and were selected by drug exposure. In fact, data obtained by SSCP analysis indicated that the IGROV-1/Pt 0.1 cells, which represent an early step during the process of selection of IGROV-1/Pt 1, carry both the wild-type and mutant p53 alleles with a wild-type predominance, thus suggesting the occurrence of a progressive selection of the mutated population operated by cisplatin. We hypothesized that SSCP and sequence analysis of IGROV-1 genomic DNA could have been unable to reveal the presence of the hypothetical fraction of p53 mutated cells because of their detection threshold. Thus, allelic-specific gene amplification, having a greater theoretical limit of sensitivity, was performed. Experiments with plasmidic DNA demonstrated that primers carrying the specific mutations at the 3' end exclusively amplified the mutant DNA, thus supporting the specificity of sequence amplification. Using wild-type primers, we obtained amplification of genomic DNA from IGROV-1 and the two cisplatin-resistant variants IGROV-1/Pt 0.1 and IGROV-1/Pt 1, as expected, based on the presence of not only mutant but also wild-type alleles detected in these cells in PCR-SSCP experiments. Mutant primers amplified all of the three cell lines and the intensity of the bands obtained from the two resistant cell systems corresponded to the amount of p53 mutations present, based on SSCP analysis. The weak band amplified from IGROV-1 indicated that mutant cells existed before drug selection. The fact that, in non-hot allele-specific PCR experiments, amplification was found only in one of four DNA extractions from IGROV-1 cell line suggests that, in independently grown cell cultures, spontaneous enrichment of p53 mutant cells can occur. Blots of the 270 PCRs confirmed the presence of the mutated clone in the other three extractions of IGROV-1 cells, at a lower level than that observed for the first extraction (0.1%). Thus, the fraction of cells containing the p53 mutations at codon 270 is variable in independently cultured cell populations. The major problem in using the labeling procedure was that, at the working primer annealing temperatures, a weak band was revealed also with genomic DNAs from cell lines with wild-type p53 sequence. With the 270 mutated primer, using higher annealing temperature, it was possible to eliminate the amplification in the negative control. This was not the case of the 282 primer, for which nonspecific annealing was consistently observed (data not shown). Such a finding, which is probably related to the nature of the primer, does not allow definitive conclusions on the preexistence of the mutation at codon 282 because the two mutations found in IGROV-1/Pt 1 cells are localized in different alleles (14) . It is unlikely that the mutations belong to distinct mutant clones because they have been selected at the same frequency in two cisplatin-resistant sublines (IGROV-1/Pt 0.5 and IGROV-1/Pt 1; Ref. 14 ). Thus, a plausible explanation of the occasional selection of resistant mutant cells produced during drug treatment is that it is the result of both selective pressure of the drug treatment and induction of an additional mutation, produced by the genotoxic stress of the drug itself. The presence of two concomitant mutations may be a favorable event allowing the emergence of mutant resistant cells. It is likely that the selection mechanism involves a reduced susceptibility to apoptosis as a consequence of inactivation of p53 gene. The capability of cisplatin to efficiently kill ovarian carcinoma cells with wild-type p53 is expected to result in a survival advantage for mutant cells. Indeed, cisplatin-induced apoptosis has been shown to be favored in cell lines carrying wild-type p53 (21) . In our model system, cisplatin-induced apoptosis has been postulated to be p53 dependent because a reduced apoptotic response of resistant p53 mutant cells is associated with loss of p53 function (14) . A further reason for the development of a resistant phenotype in p53 mutant cells is that loss of p53 function contributes to genomic instability as a consequence of defects in checkpoint pathways and gain of novel functions, including overexpression of defense factors (22) . This interpretation is consistent with a preliminary observation that the introduction of a mutant p53 (codon 282) in wild-type IGROV-1 cells conferred a low level of resistance to cisplatin (data not shown).

Despite the technical difficulty to document the sequence of the events involved in the development of this type of resistance (i.e., p53-mediated), our model provides indirect evidence that selection of spontaneous or induced mutant cells is a process involved in the development of clinical resistance. A high degree of resistance to DNA-damaging agents may arise readily after short periods of treatment or gradually as a result of treatment over prolonged periods. The known tumor heterogeneity in terms of p53 mutations may account for the pattern of response of ovarian carcinoma to conventional treatments, based on DNA-damaging agents (i.e., cisplatin and alkylating agents; Ref. 16 ). Indeed, intrinsic drug resistance to cisplatin-based first line therapy was found to be correlated with missense mutations of p53 gene (16) . In partial responses, resistance may arise quickly and relatively easily if mutant resistant cells constitute an appreciable fraction of tumor cell population at the time of therapy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines and Culture Conditions.
The IGROV-1 ovarian carcinoma cell line originally obtained from Dr. J. Benard (Institut Gustave Roussy, Villejuif, France) was cultured in RPMI 1640 with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD). The cisplatin-resistant variant IGROV-1/Pt 1 was generated by continuous exposure of the IGROV-1 cell line to increasing concentration of cisplatin starting from 0.1 up to 1 µg/ml (23) . IGROV-1/Pt 0.1 cells represents an early passage during the process of selection of IGROV-1/Pt 1 cells and were cultured in 0.1 µg/ml cisplatin.

Cytotoxicity Studies.
Cytotoxicity after a 96-h exposure to cisplatin was assessed by tetrazolium dye (MTT) assay (24) . This antiproliferative assay was used because the colony-forming assay was not suitable in our cell system as a consequence of a low plating efficiency. However, a preliminary evaluation of the cytotoxic effects using the colony forming assay supports a comparable resistance index of the IGROV-1/Pt 1 found with the MTT method. Preliminary experiments were performed to determine the appropriate seeding number of cells (4000 cells/well) after confirming the linear relationship between the absorbance and the number of cells in the growth curve of each cell line. The IC50 is defined as the inhibitory drug concentration causing a 50% reduction of A550 nm(decrease of cell growth) over that of untreated control.

Allele-specific DNA Amplification.
Allele-specific DNA amplification was carried out in PCR experiments to amplify 0.35 µg of genomic DNA extracted from IGROV-1, IGROV-1/Pt 1, and IGROV-1/Pt 0.1 cell lines, according to standard techniques (25) . For IGROV-1 cells, four independent DNA extractions (from cells at comparable passages but from different thawings, named I, II, III, and IV) of the original cell line used to select the IGROV-1/Pt 1 subline were used. Primers carrying the desired mutations (T->A codon 270, C->T codon 282; Table 1Citation ) in the 3' terminus and the corresponding wild-type primers were used. The oligonucleotide that was complementary to primers p53-270m or p53-270w was the same as that used for PCR-SSCP analysis (p53-8.3; amplified band, 193 bp), whereas that used with primers p53-282m and p53-282w was 7 nucleotides upstream [designated p53–8.3(-7); amplified band, 152 bp] for matching the melting temperature. All of the reactions were performed with KlenTaq1 DNA polymerase (BioNova s.r.l., Bologna, Italy), an enzyme characterized by a high heat stability, in a volume of 50 µl containing template DNA, primers (0.5 µM each), dNTPs (200 µM each), and reaction buffer. The mutant allele was specifically amplified at the annealing temperature at which no amplification was revealed with genomic DNA from cell lines used as negative controls (H460, A549, and N592). For the 270 mutation, reactions were carried out for 30 cycles at 94°C for 1 min, 68°C (58°C for the wild-type allele) for 40 s, and 72°C for 1 min. For the 282 mutation, reactions were: 94°C for 1 min, 72°C (62°C for the wild-type allele) for 30 s, and 72°C for 1 min (30 cycles). An aliquot (25 µl) of each PCR was run in a 2% agarose gel, transferred to nitrocellulose membrane, and hybridized with the corresponding radiolabeled PCR mutated fragment from IGROV-1/Pt 1 (Megaprime DNA labeling system, Amersham, Little Chalfont, United Kingdom). To estimate the proportion of mutant alleles, genomic DNA from IGROV-1/Pt 1 was mixed at various ratios with genomic DNA from wild-type p53 cells (from 1:10 to 1:10000) and subjected to allele-specific PCR. All of the experiments were repeated at least three times.


View this table:
[in this window]
[in a new window]
 
Table 1 Allele-specific primers and primers used for mutagenesisa

 
Allele-specific PCR analysis was used for amplification of plasmid DNA. Ten ng of the pC53 plasmid carrying wild-type (pC53-SN3) or mutated (pC53-M) sequences were amplified using the AmpliTaq DNA polymerase (Perkin-Elmer Corp., Branchburg, NJ) with the same PCR condition described above. The downstream primer (p53–6As) was specific for cDNA sequence and was used with all of the upstream primers (expected bands of 281 bp for 270 mutation and 245 bp for the 282 mutation).

SSCP and Sequencing Analysis.
SSCP analysis for the detection of p53 gene mutation has been described previously (26) . Primers used to amplify exon 8 were p53-8.3 and p53-8.5 (Table 1)Citation . PCR-amplified exons were subjected to direct DNA sequencing with an AmpliCycle Sequencing kit (Perkin-Elmer Corp.). To sequence p53 cDNAs inserted into pC53 plasmids, we used primer 5S.

Plasmid Mutagenesis.
The pC53-SN3 plasmid (27) carrying wild-type p53 cDNA was mutagenized at sites 270 and 282 using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA). Briefly, for each site, two synthetic oligonucleotide primers containing the desired mutation (p53-270As/p53-270S and p53-282As/p53-282S; Table 1Citation ) were used to amplify the plasmid template with Pfu DNA polymerase, which replicates both plasmid strands with high fidelity and without displacing the mutant primer. The PCR product was then treated with the Dpn I endonuclease to digest the parental DNA template and to select for mutation-containing synthesized DNA. The nicked vector incorporating the mutation was then transformed into Epicurian Coli XL-1-Blue supercompetent cells, and mutagenized plasmids were subjected to direct sequencing.


    Acknowledgments
 
We thank Dr. M. Asada for helpful discussion and Laura Zanesi for editorial assistance.


    Footnotes
 
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 This work was partially supported by the Consiglio Nazionale delle Ricerche, Finalized Project ACRO, (Rome), by the Associazione Italiana per la Ricerca sul Cancro (Milan) and by the Ministero della Sanità (Rome). Back

2 To whom requests for reprints should be addressed, at Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390267; Fax: 39-02-2390692; E-mail: zunino{at}istitutotumori.mi.it. Back

3 The abbreviations used are: SSCP, single-strand conformation polymorphism; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back

Received for publication 2/ 4/99. Revision received 4/29/99. Accepted for publication 5/24/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Weiss R. B., Christian M. C. New cisplatin analogues in development: A review. Drugs, 46: 360-377, 1993.[Medline]
  2. Thigpen T., Vance R., Puneky L., Khansur T. Chemotherapy in advanced ovarian carcinoma: current standards of care based on randomized trials. Gynecol. Oncol., 55: 97-107, 1994.
  3. Kelland L. R. The molecular basis of cisplatin sensitivity/resistance. Eur. J. Cancer, 30A: 725-727, 1994.
  4. Chu G. Cellular responses to cisplatin. J. Biol. Chem., 269: 787-790, 1994.[Abstract/Free Full Text]
  5. Dancey J., Le Chevalier T. Non-small cell lung cancer: an overview of current management. Eur. J. Cancer, 33: S2-S7, 1997.
  6. Zunino F., Perego P., Pilotti S., Pratesi G., Supino R., Arcamone F. Role of apoptotic response in cellular resistance to cytotoxic agents. Pharmacol. Ther., 76: 177-185, 1997.[Medline]
  7. Hickman J. A. Apoptosis and chemotherapy resistance. Eur. J. Cancer, 32A: 921-926, 1996.
  8. Mowat M. R. A. p53 in tumor progression: life, death and everything. Adv. Cancer Res., 74: 25-48, 1998.[Medline]
  9. Steele R. J., Thompson A. M., Hall P. A., Lane D. P. The p53 tumor suppressor gene. Br. J. Surg., 85: 1460-1467, 1998.[Medline]
  10. Hartmann A., Blaszyk H., Kovach J. S., Sommer S. S. The molecular epidemiology of p53 gene mutations in human breast cancer. Trends Genet., 13: 27-33, 1997.[Medline]
  11. Eliopoulos A. G., Kerr D. J., Herod J., Hodgking L., Krajewski S., Reed J. C., Young L. S. The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and Bcl-2. Oncogene, 11: 1217-1228, 1995.[Medline]
  12. Milner J. DNA damage, p53 and anticancer therapies. Nat. Med., 1: 879-880, 1995.[Medline]
  13. Fan S., El-Deiry W. S., Bae I., Freeman J., Jondle D., Bathia K., Fornace A. J., Magrath I., Kohn K. W., O’Connor M. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA-damaging agents. Cancer Res., 54: 5824-5830, 1994.[Abstract/Free Full Text]
  14. Perego P., Giarola M., Righetti S. C., Supino R., Caserini C., Delia D., Pierotti M. A., Miyashita T., Reed J. C., Zunino F. Association between cisplatin resistance and mutation of p53 gene and reduced Bax expression in ovarian carcinoma cell systems. Cancer Res., 56: 556-562, 1996.[Abstract/Free Full Text]
  15. Gallagher W. M., Cairney M., Schott B., Roninson I. B., Brown R. Identification of p53 genetic suppressor elements which confer resistance to cisplatin. Oncogene, 14: 185-193, 1997.[Medline]
  16. Righetti S. C., Della Torre G., Pilotti S., Mènard S., Ottone F., Colnaghi M. I., Pierotti M. A., Lavarino C., Cornarotti M., Oriana S., Böhm S., Bresciani G. L., Spatti G., Zunino F. A comparative study of p53 gene mutations, protein accumulation and response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Res., 56: 689-693, 1996.[Abstract/Free Full Text]
  17. Perego P., Righetti S. C., Supino R., Delia D., Caserini C., Carenini N., Bedogne B., Broome E., Krajewski S., Reed J. C., Zunino F. Role of apoptosis and apoptosis-related proteins in the cisplatin-resistant phenotype of human tumor cell lines. Apoptosis, 2: 540-548, 1997.[Medline]
  18. Harris C. C. Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J. Natl. Cancer Inst., 88: 1442-1455, 1996.[Abstract/Free Full Text]
  19. Kirsch D. G., Kastan M. B. Tumor suppressor p53: implications for tumor development and prognosis. J. Clin. Oncol., 16: 3158-3168, 1998.[Abstract/Free Full Text]
  20. Goldie J. H., Coldman A. J. A mathematic model for relating the drug sensitivity of tumours to their spontaneous mutation rate. Cancer Treat. Rep., 63: 1727-1733, 1979.[Medline]
  21. O’Connor P. M., Jackman J., Bae I., Myers T. G., Fan S., Mutoh M., Scudiero D. A., Monks A., Sausville E. A., Weinstein J. N., Friend S., Fornace A. J., Jr., Kohn K. W. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlation with the growth-inhibitory potency of 123 anticancer agents. Cancer Res., 57: 4285-4300, 1997.[Abstract/Free Full Text]
  22. Harris C. C. Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J. Natl. Cancer Inst., 88: 1442-1455, 1996.
  23. Perego P., Romanelli S., Carenini N., Magnani I., Leone R., Bonetti A., Paolicchi A., Zunino F. Ovarian cancer cisplatin-resistant cell lines: multiple changes including collateral sensitivity to taxol. Ann. Oncol., 9: 423-430, 1998.[Abstract/Free Full Text]
  24. Alley M. C., Scudiero D. A., Monks A., Hursey M. L., Czerwinski M. J., Fine D. L., Abbott B. J., Mayo J. G., Shoemaker R. H., Boyd M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res., 48: 589-601, 1988.[Abstract/Free Full Text]
  25. Sambrook J., Fritsch E. F., Maniatis T. Molecular cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY 1989.
  26. Donghi R., Longoni A., Pilotti S., Michieli P., Della Porta G., Pierotti M. A. Gene p53 mutations are restricted to poorly differentiated and undifferentiated carcinomas of the thyroid gland. J. Clin. Invest., 91: 1753-1760, 1993.
  27. Baker S. J., Markowitz S., Fearon E. R., Willson J. K. V., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science (Washington DC), 249: 912-915, 1990.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
Clin. Cancer Res.Home page
J. Carr-Wilkinson, K. O'Toole, K. M. Wood, C. C. Challen, A. G. Baker, J. R. Board, L. Evans, M. Cole, N.-K. V. Cheung, J. Boos, et al.
High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma
Clin. Cancer Res., February 15, 2010; 16(4): 1108 - 1118.
[Abstract] [Full Text] [PDF]


Home page
Clin. Cancer Res.Home page
D. Mezzanzanica, E. Balladore, F. Turatti, E. Luison, P. Alberti, M. Bagnoli, M. Figini, A. Mazzoni, F. Raspagliesi, M. Oggionni, et al.
CD95-Mediated Apoptosis Is Impaired at Receptor Level by Cellular FLICE-Inhibitory Protein (Long Form) in Wild-Type p53 Human Ovarian Carcinoma
Clin. Cancer Res., August 1, 2004; 10(15): 5202 - 5214.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
K. Szepeshazi, A. V. Schally, G. Halmos, P. Armatis, F. Hebert, B. Sun, A. Feil, H. Kiaris, and A. Nagy
Targeted Cytotoxic Somatostatin Analogue AN-238 Inhibits Somatostatin Receptor-positive Experimental Colon Cancers Independently of Their p53 Status
Cancer Res., February 1, 2002; 62(3): 781 - 788.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
N. Keshelava, J. J. Zuo, P. Chen, S. N. Waidyaratne, M. C. Luna, C. J. Gomer, T. J. Triche, and C. P. Reynolds
Loss of p53 Function Confers High-Level Multidrug Resistance in Neuroblastoma Cell Lines
Cancer Res., August 1, 2001; 61(16): 6185 - 6193.
[Abstract] [Full Text] [PDF]


Home page
Clin. Cancer Res.Home page
M. S. Rieber, U. Zangemeister-Wittke, and M. Rieber
p53-independent Induction of Apoptosis in Human Melanoma Cells by a bcl-2/bcl-xL Bispecific Antisense Oligonucleotide
Clin. Cancer Res., May 1, 2001; 7(5): 1446 - 1451.
[Abstract] [Full Text]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Righetti, S. C.
Right arrow Articles by Zunino, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Righetti, S. C.
Right arrow Articles by Zunino, F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation