A novel trans-platinum coordination complex possessing in vitro and in vivo antitumor activity

As part of a drug discovery program to discover more effective platinum-based anticancer drugs, a series of platinum complexes of trans coordination geometry centered on trans-ammine(cyclohexylaminedichlorodihydroxo)platinum(IV) (JM335) has been evaluated in vitro against a panel of cisplatin-sensitive and cisplatin-resistant human tumor cell lines (predominantly ovarian). In vitro, against 5 human ovarian carcinoma cell lines, JM335 was comparably cytotoxic to cisplatin itself and over 50-fold more potent than transplatin (mean 50% inhibitory concentrations: JM335, 3.1 microM; cisplatin, 4.1 microM; transplatin, 162 microM). With the use of seven pairs of human tumor cell lines (parent and subline with acquired resistance to cisplatin and encompassing all of the known major mechanisms of resistance to cisplatin) JM335 exhibited a different cross-resistance pattern to that of its cis isomer (JM149). JM335 showed non-cross-resistance in six of the seven resistant lines, cross-resistance in the A2780cisR line possibly being associated with high levels of glutathione. Preliminary intracellular DNA binding studies showed that in contrast to transplatin, JM335 was efficient at forming DNA-DNA interstrand cross-links. In vivo, JM335 produced growth delays in excess of 15 days against 4 of 6 human ovarian carcinoma xenografts and was unique among the complexes studied in retaining some efficacy against a cisplatin-resistant subline of the murine ADJ/PC6 plasmacytoma. JM335 is the first trans-platinum complex to demonstrate marked antitumor efficacy against both murine and human s.c. tumor models and represents a significant structural lead to complexes capable of circumventing cross-resistance to cisplatin.     

The Canadian health care crisis and how to survive it: can cost effectiveness save Canadian doctors?

Patients with head and neck squamous cell carcinoma (HNSCC) treated with cisplatin show a large inter-individual variation in tumor response. Little is known about factors that contribute to this variation. The aim of our study was to correlate the sensitivity to cisplatin with a number of cellular parameters using a panel of 10 human HNSCC cell lines. A 7-fold variation in response after 72 hr of exposure to cisplatin as determined in a colorimetric proliferation assay was observed. The IC50 values did not correlate with the DNA index, the cellular doubling time or the expression of differentiation markers. Intracellular platinum (Pt) concentrations were measured by atomic absorption spectroscopy after exposing the cells to 10 microM cisplatin for 1-72 hr. The intracellular Pt levels increased up to 24 hr. One cell line, derived from the tumor of a patient previously treated with radiotherapy, accumulated much more Pt than the other cell lines. For these other cell lines, a significant positive correlation was found between Pt accumulation and sensitivity. In conclusion, cisplatin-induced growth inhibition in HNSCC in vitro is generally positively correlated with cellular Pt levels. However, the fact that occasionally cancer cells can survive despite high intracellular Pt levels indicates that additional parameters are needed to explain a response unequivocally.     

Characterisation of the unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines of the SuSa testicular teratoma cell line

Three etoposide-selected resistant sublines of the SuSa testicular teratoma cell line expressing 9-, 21- and 33-fold levels of resistance, proved increasingly cross resistant to cisplatin with levels approximating to 3-, 4- and 6-fold in sublines VPC2, VPC3 and VPC4, respectively. Cisplatin resistance was not associated with any significant modifications in levels of total glutathione or associated enzyme activities. Decreased platinum (Pt) accumulation was detected, although this did not correlate either with total platination levels judged immunochemically or with peak induction of interstrand crosslinks (ISC) determined by alkaline elution. Following exposure to cisplatin in the least resistant subline, VPC2, total platination levels were markedly decreased (3-fold) relative to those of the parental cells, whilst peak ISC levels were markedly increased (4-fold). In the most highly resistant subline, VPC4, peak levels of ISCs were even higher (9-fold), although total platination levels remained comparable with those in parental cells. Both VPC2 and VPC4 cells appeared highly proficient in removing ISCs, unlike the parental cells. However, whilst VPC2 cells appeared to share deficient removal of the intrastrand platinated lesions with parental cells, VPC4 cells proved proficient in removing specific adducts in the sequence pApG. This unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines derived from an inherently repair deficient parental cell line, SuSa, therefore appears to be associated with enhanced removal of the specific intrastrand crosslinks in the sequence pApG and/or of DNA-DNA ISCs. Similar mechanisms have been implicated in two other cisplatin resistant SuSa sublines selected following in vitro exposure to the drug itself or to fractionated X-irradiation.     

Lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in testicular germ cell tumour cell lines

We investigated the role of p53 and of the Bcl-2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin-induced apoptosis. In contrast to wild-type p53-containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with non-functional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells failed to undergo cisplatin-induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4-fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53-independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in drug-induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin-induced apoptotic pathway in TGCT cell lines might be p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat.     

Cellular and molecular determinants of cisplatin resistance

Cisplatin and carboplatin are among the most active and widely used cytotoxic anticancer drugs. However, the acquisition or presence of resistance significantly undermines the curative potential of these drugs against many malignancies. Multiple potential mechanisms of resistance have been identified at the cellular and molecular levels. Alterations in cellular pharmacology, including decreased drug accumulation, increased cellular thiol levels and increased repair of platinum-DNA damage, have been observed in numerous model systems. More recently, it has become apparent that an enhanced capacity to tolerate cisplatin-induced damage may also contribute to resistance. Alterations in proteins that recognise cisplatin-DNA damage (mismatch repair and high-mobility group (HMG) family proteins) and in pathways that determine sensitivity to apoptosis may contribute to damage tolerance. It remains to be determined whether any of these mechanisms contribute significantly to resistance in the clinical setting. Ongoing biochemical modulation and translational correlative trials should clarify which specific mechanisms are most relevant to clinical cisplatin resistance. Such investigations have the potential to improve the ability to predict likelihood of response and should identify potential targets for pharmacological or molecular intervention.     

Cell biological markers of drug resistance in ovarian carcinoma

The aim of the study is to review the mechanisms of resistance to four classes of drugs that are widely used in ovarian carcinoma: platinum (cisplatin/carboplatin) compounds, classical alkylating agents (cyclophosphamide/melphalan), natural drugs (doxorubicin), and "new drugs" (taxol and taxotere). Both platinum and classical alkylating agents mediate their cytotoxicity by the formation of drug-DNA adducts, resulting in DNA damage. Therefore, drug resistance mechanisms are (in part) comparable. In ovarian carcinoma cell lines increased repair of DNA damage and increased detoxification by binding of drugs to glutathione, possibly catalyzed by glutathione S-transferases, have been identified as the most prominent resistance mechanisms to these drugs. Studies on the role of DNA repair mechanisms and glutathione in human ovarian carcinoma are hampered by the complexity of enzyme systems involved in DNA repair and intratumor heterogeneity for glutathione. Resistance to doxorubicin appears to be mediated by enhanced efflux from the cell by increased expression of membrane glycoproteins acting as a drug efflux pump, such as P-glycoprotein. Resistance to doxorubicin can also be due to quantitative and/or qualitative changes in the nuclear target of doxorubicin, topisomerase (Topo) II. Finally, resistance to taxol may be mediated by enhanced expression of P-glycoprotein, while presumed other mechanisms such as alterations in tubulin structure, the cellular "target" of taxol, and changes in polymerization of tubulin are still largely unresolved. Several ways to modulate the reviewed resistance mechanisms are also described. In conclusion, this review shows that many cell biological factors may be involved in drug resistance. The relevance of the identification of most of these factors in ovarian carcinoma patients however remains to be established.     

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells

Overexpression of the erbB-2 tyrosine kinase receptor, p185erbB-2, is a common alteration in non-small cell lung cancer (NSCLC) and has been associated with poor prognosis and a tumor drug resistance phenotype. In this study, we have examined the consequences of erbB-2 depletion on DNA repair, cell cycle, and apoptosis using a panel of NSCLC cell lines constitutively overexpressing erbB-2 receptor. Depletion of the erbB-2 was achieved using the tyrosine kinase inhibitor CP127,374 which promotes erbB-2 degradation. Treatment with CP127,374 concentrations which deplete erbB-2 and inhibit tyrosine phosphorylation resulted in downregulation of DNA repair mechanisms and cell accumulation at G1 phase of the cell cycle. GI arrest was observed in cells with mutated p53 as well as cells lacking p53 protein, suggesting a p53-independent mechanisms. NSCLC cells which overexpress erbB-2 were more resistant to cisplatin-induced cytotoxicity in comparison to cells expressing low levels of erbB-2. Treatment with CP127,374 alone did not result in any induction of apoptosis. A combination of CP127,374 and cisplatin, however, was more potent in cell growth inhibition and induction of apoptosis compared to treatment with cisplatin alone. Together, our results further support a pivotal role of erbB-2 signaling in the regulatory balance between DNA repair, cell cycle checkpoints and apoptosis; all these mechanisms are essential determinants for tumor cell destiny following chemotherapy stress.     

Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.     

Fas-mediated apoptosis with normal expression of bcl-2 and p53 in lymphocytes from aplastic anaemia

The p53 tumor suppressor gene is critical in regulating cell proliferation following DNA damage, and disruption of p53 protein function by mutation has been implicated as a factor responsible for resistance of tumor cells to chemotherapeutic agents. Our studies were initiated by asking whether the translational product of the p53 gene is associated with cisplatin resistance in the 2780CP human ovarian tumor model. We have demonstrated by single-strand conformation polymorphism analysis and sequencing that p53 in parental cisplatin-sensitive A2780 cells was wild type. In 2780CP cells, however, a mutation was found in exon 5 at codon 172 (Val to Phe). Interestingly, exposure to X-rays resulted in p53 induction in both A2780 and 2780CP tumor models. The p53 increases by the ionizing radiation were accompanied by concomitant increases in levels of the p53-regulated p21Waf1/Cip1 protein and led to arrest of cells in G1 phase of the cell cycle. A yeast functional assay confirmed that p53 in A2780 was wild type, but, more importantly, it provided evidence that the p53 mutation in 2780CP cells was temperature sensitive and heterozygous. These experiments demonstrate that sensitive and resistant cells have normal p53 functions, despite the presence of p53 mutation in the 2780CP model. In parallel investigations using the Western technique, exposure of A2780 cells to clinically relevant concentrations of cisplatin (1-20 microM) resulted in time- and dose-dependent increases in p53, together with coordinate increases in p21Waf1/Cip1. In contrast, cisplatin did not induce these proteins in 2780CP cells to any significant degree. The results indicate that a defect exists in the signal transduction pathway for p53 induction following cisplatin-induced DNA damage in 2780CP cells, and this may represent a significant mechanism of cisplatin resistance. Furthermore, induction of p53 in 2780CP cells by X-rays, but not cisplatin, strongly suggests that independent pathways are involved in p53 regulation for the two DNA-damaging agents.     

Cytotoxicity and tolerance to DNA adducts of alicyclic mixed amine platinum(II) homologs in tumor models sensitive and resistant to cisplatin or tetraplatin

Structure-cytotoxicity relationships for six alicyclic cis-(NH3)(R-NH2)Cl2Pt(II) complexes, where R=C3H5, C4H7, C5H9, C6H11, C7H13 and C8H15 (complexes abbreviated C3, C4, C5, C6, C7 and C8, respectively), were evaluated against four sensitive (L1210/0, A2780, FSaIIC and Colon 26), two cisplatin-resistant (L1210/DDP and 2780CP) and two tetraplatin-resistant (L1210/DACH and 2780TP) murine and human tumor cell lines. The studies demonstrated that in general the structure of C6 was optimal within the homologous series for cytotoxic potency against these tumor models. Biochemical pharmacologic studies indicated that the greater sensitivity of cells to C6 could be correlated with their low tolerance to DNA damage induced by this homolog. These results provide evidence for the alicyclic ring size as a structural determinant of DNA damage tolerance and anti-tumor activity in sensitive and resistant tumor cells.     

ATP-dependent glutathione conjugate export pump

KCP4 cells are resistant to cisplatin and have a GS-X pump different from MRP. The GS-X pump was suggested to be involved in reducing the accumulation of cisplatin in KCP-4 cells. The expression of cMOAT was 4-to 6-fold higher in KCP-4 cells and two other cisplatin-resistant human cell lines. It is still not clear whether the cisplatin resistance in KCP-4 cells are attributed to cMOAT. Other members of the MRP/GS-X pump family have been reported. Amino acid sequence of EBCR of rabbit is 91% identical to that of human cMOAT. On the view of cancer chemotherapy, it is very important to understand the structure and function of GS-X pumps since they may be involved in not only drug resistance but also drug metabolism and side effects.     

Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway

Cisplatin resistance, induced in murine fibrosarcoma cells (SSK) in vitro or in vivo by low-dose irradiation, can be overcome by activation of the cyclic GMP(cGMP)-dependent transduction pathway. This is mediated either by stimulating cGMP formation with sodium nitroprusside or by replacing cGMP with a selective activator of the cGMP-dependent protein kinase, 8-bromo-cGMP. The cyclic AMP-dependent transduction pathway is not involved in cisplatin resistance. Instead, activation of cAMP sensitises both parental and resistant SSK cells equally to the action of cisplatin. There is a 1.8 to 2.5-fold increase in drug toxicity, depending on the activating agent. Enhancement of cisplatin sensitivity is induced by specific inhibition of cAMP hydrolysis, increase in cAMP formation or by increasing the activation potential to cAMP-dependent protein kinase by specific cAMP analogues. Cells that have lost cisplatin resistance respond to cGMP- or cAMP-elevating agents in the same way as the parental SSK cells. The radiation sensitivity is unchanged in all cell lines, even after activation of cAMP or cGMP. These results suggest that specific DNA repair pathways are altered by radiation but affected only in cisplatin damage repair, which is regulated by cGMP. Although there is ample cooperativity and interaction between the cAMP- and the cGMP-dependent transduction pathways, specific substrate binding by cGMP appears to play an important role in radiation-induced cisplatin resistance.     

Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning

Camptothecins are a new class of anticancer drugs that target DNA topoisomerase I; current efforts are directed toward elucidating optimal combinations of these drugs with other antineoplastic agents. A rationale for the use of sequential therapy involving the combination of camptothecins with topoisomerase II-targeting drugs, such as etoposide, has arisen from observations of increased topoisomerase II protein levels in cell lines resistant to camptothecin. In an effort to understand potential mechanisms of resistance to this strategy, we developed a U-937 cell subline, denoted RERC, that is capable of surviving exposure to sequential topoisomerase poisoning. The RERC cells are 200-fold resistant to camptothecin, 8-fold resistant to etoposide, and 10-fold hypersensitive to cisplatin compared to the parental U-937 cells. Biochemical analyses indicate that the resistant phenotype involves alterations in both topoisomerase I and topoisomerase IIalpha. Topoisomerase I catalytic activity in the resistant cells is similar to that of the parental line but is resistant to camptothecin. Moreover, the resistant cells express a single mRNA species of topoisomerase I that codes for a mutation in codon 533. In addition, topoisomerase IIalpha protein levels are decreased 10-fold in the resistant line, coincident with a two-fold decrease in the expression of topoisomerase IIalpha mRNA. Collectively, these results indicate that resistance to sequential topoisomerase poisoning may involve a reduction in total cellular topoisomerase activity.     

Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin

The c-Abl nonreceptor tyrosine kinase and the c-Jun NH2-terminal kinase (JNK/stress-activated protein kinase) are activated during the injury response to the DNA-damaging agent cisplatin. Loss of DNA mismatch repair activity results in resistance to cisplatin in human cancer cells, suggesting that the mismatch repair proteins function as a detector for cisplatin DNA adducts. To identify signaling pathways activated by this detector, we investigated the effect of the loss of DNA mismatch repair function on the ability of cisplatin to activate the JNK and c-Abl kinases. The results demonstrate that cisplatin activates JNK kinase 3.8 +/- 0.2-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that activation of c-Abl is completely absent in the DNA mismatch repair-deficient cells. Furthermore, the results show that cisplatin-induced activation of JNK occurs through a stress-activated protein kinase/extracellular signal-regulated kinase kinase 1-independent mechanism. We conclude that activation of JNK and c-Abl by cisplatin is in part dependent upon the integrity of DNA mismatch repair function, suggesting that these kinases are part of the signal transduction pathway activated when mismatch repair proteins recognize cisplatin adducts in DNA.     

Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis

Malignant glioblastomas grow very rapidly and are generally resistant to either DNA-damaging drugs or gamma-irradiation. If tumor cells could be made more susceptible to cell death with treatments, this would clearly represent a significant improvement in the success of treatment. Recently, telomerase has become a focus of interest among oncologists as a target for treating cancer cells. Telomerase elongates telomeric DNA repeats (TTAGGG)n and is important in protecting and replicating DNA. The vast majority of tumor cells, indeed, express telomerase activity whereas normal somatic cells, except for a few cells, do not. Since telomerase is essential for protecting DNA, we may be able to make tumors more sensitive to treatments with DNA-damaging drugs by inhibiting telomerase activity. In this study, we used cis-diamminedichloroplatinum (cisplatin)-sensitive U87-MG cells and cisplatin-resistant U251-MG of human malignant glioblastoma cell lines. U87-MG cells did not express telomerase activity, whereas telomerase was highly detected in U251-MG cells. Interestingly, inhibition of telomerase with an antisense telomerase expression vector not only decreased telomerase activity but also increased susceptibility to cisplatin-induced apoptotic cell death in U251-MG cells. These findings suggest that treatment with antisense telomerase may represent a new chemosensitisation for tumors resistant to anticancer drugs.     

Retinoic acids induce growth inhibition and apoptosis in adult T-cell leukemia (ATL) cell lines

In order to elucidate the mechanisms of cisplatin (cis-diamminedichloroplatinum; CDDP)-resistant tumor cells, we previously established a CDDP-resistant KB cell line (KBrc cells) from a parental KB cell line derived from epidermoid carcinoma (KB cells). The KBrc cells were resistant to 5 kinds of platinum (Pt) drugs. Intracellular Pt concentrations in KBrc cells were lower than in KB cells. Decrease of intracellular Pt concentrations was one of the CDDP-resistant mechanisms. When we measured changes of intracellular calcium ion concentration ([Ca2+]i) during exposure to high-dose CDDP, a sustained elevation of the [Ca2+]i level was observed in the KB cells. These results suggest that the mechanisms underlying CDDP resistance involve changes in calcium channels and an alteration of calcium homeostasis in the tumor cell line.     

Apoptosis and cancer chemotherapy

Cytotoxic drugs currently remain as the basis for the chemotherapy of metastatic cancer. Why they fail to kill sufficient tumour cells in the major human solid cancers, such as the carcinomas, is suggested in this review to be due to the inherent inability of these cells to engage apoptosis after drug-induced damage. As a paradigm for drug resistant cancers, the resistance of bladder carcinoma cell lines to DNA damaging drugs is described here in terms of their response to the topoisomerase II poison etoposide. 60%-70% of bladder carcinomas have mutant p53; this can prevent the detection of and response to DNA damage. In vitro studies with a bladder carcinoma cell line containing a wild type p53 showed that it underwent a G1 checkpoint after etoposide, potentially allowing DNA damage repair, as well as apoptosis. In lines with mutant or non-functional p53 there is no checkpoint and no apoptosis. All lines showed constitutive expression of bcl-2 and bcl-XL (the suppressors of apoptosis) with low and non-inducible levels of bax (a promoter of apoptosis). Taken together, this menu of gene expression is more favourable to survival than apoptosis after the imposition of drug-induced DNA damage and may contribute to their inherent drug resistance.     

Evidence of cisplatin-induced senescent-like growth arrest in nasopharyngeal carcinoma cells

Cellular senescence is a programmed cell response leading to growth arrest in human diploid fibroblasts. We have shown that a nasopharyngeal carcinoma cell line, CNE1, following treatment by the DNA-damaging agent cisplatin, can undergo cellular senescent-like growth arrest, similar to fibroblasts, judged by cellular morphological changes and the expression of senescence-associated beta-galactosidase (SA-beta-gal). This senescent-like change was dose related; at 0.5 microgram/ml, the percentage of cisplatin-induced SA-beta-gal-positive cells was high (40-96%), and the staining was intense. Higher doses (1.0 and 2.0 micrograms/ml) of cisplatin induced lower SA-beta-gal expression (30-70%), and the process was irreversible. This cisplatin-induced cellular senescent-like response was not due to the inhibition of telomerase activity. Our results indicate that cellular senescent-like pathways exist in nasopharyngeal carcinoma cells and can be induced by cisplatin. Our evidence suggests that cellular senescent-like responses may be a cellular protection mechanism that acts differently in response to different degrees of cellular damage.     

Contribution of the cyclin-dependent kinase inhibitor p27KIP1 to the confluence-dependent resistance of HT29 human colon carcinoma cells

We have previously shown that growth of HT29 human colorectal cancer cells at confluence increased their resistance to the cytotoxic agent cisplatin. This study further explores the mechanisms of this resistance phenotype. DNA platination induced by cisplatin exposure is slightly reduced by confluence. However, at an equivalent DNA platination level, non-confluent cells accumulate in the G2/M phase of the cell cycle, demonstrate aberrant mitotic figures and die by apoptosis, while confluent cells progress slowly through the cell cycle, do not reach mitosis and are more resistant to drug-induced cell death. At a molecular level, cisplatin enhances cyclin B and p34cdc2 levels and histone H1 kinase activity in non-confluent, but not in confluent, cells. Furthermore, when HT29 cells reach confluence, expression of the cyclin-dependent kinase inhibitor p27Kip1 increases and cells accumulate in the G0/G1 phase of the cell cycle. Transfection-mediated over-expression of p27Kip1 in non-confluent HT29 cells decreases the cytotoxic activity of cisplatin as well as its ability to trigger apoptosis. Non-confluent HT29 cells over-expressing p27Kip1 are also more resistant to doxorubicin, etoposide and 5-fluorouracil. Our results suggest that p27Kip1 contributes to the confluence-dependent resistance phenotype.