Autoregulation of topoisomerase I expression by supercoiling sensitive transcription.

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.

An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene.

We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 µM) and 72.9% (8 µM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 µM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria.

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines.

BACKGROUND: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. METHODS: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. RESULTS: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. CONCLUSIONS: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.

A simplified protocol for high-yield expression and purification of bacterial topoisomerase I.

Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity.

Diagnostic criteria of systemic sclerosis.

Systemic sclerosis (SSc) is a multisystem disease characterized by vascular abnormalities, immune system activation manifested by SSc-specific autoantibodies and disturbances in fibroblast function. The clinical manifestations are highly heterogeneous and commonly include skin thickening, Raynaud's phenomenon, digital ulcers, gastroesophageal reflux disease, interstitial lung disease and cardiac diastolic dysfunction. The diagnosis of SSc in a patient with typical end-organ disease is relatively straight-forward, but is unsatisfactory because it implies that the diagnosis is delayed until irreversible tissue damage is present. Diagnostic criteria are generally designed to facilitate the clinical process and to allow early institution of therapy to relieve symptoms and possibly prevent irreversible damage. Several attempts at defining diagnostic criteria for SSc have been made in the past. Raynaud's phenomenon, SSc-specific autoantibodies and nailfold capillary abnormalities are among the most promising items likely to be retained in a final set of diagnostic criteria. The EULAR Scleroderma Trial and Research group (EUSTAR) is currently in the process of prospectively validating a set of diagnostic criteria for the very early diagnosis of SSc and results are expected in 2015.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico.

BACKGROUND: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear. METHODS: The three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation. RESULTS: The three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine. CONCLUSIONS: These results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells.

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.

Topoisomerase I expression correlates to response to neoadjuvant irinotecan-based chemoradiation in rectal cancer.

Biomarkers may help predict the efficacy of neoadjuvant chemoradiation in patients with rectal cancer. We hypothesized that the expression of topoisomerase I (Topo I) and thymidylate synthase (TS) may help predict the treatment response in patients undergoing irinotecan and capecitabine-based chemoradiation. Patients with rectal cancer (cT3/4Nx or Tx/N+) received neoadjuvant chemoradiotherapy within clinical studies with irinotecan and capecitabine. Samples of normal and tumour tissues were collected before the start of the treatment and during surgical resection. Topo I and TS were measured using real-time PCR. The results of gene expression levels were compared between responders (defined as ypT0-2 ypN0) and nonresponders (ypT3-4 or ypN1/2). A total of 38 patients were analysed, 18 of them were responders. The biopsies of the untreated tumour tissue of responding patients showed a significant higher expression of Topo I compared with nonresponding patients (P = 0.015). Normal tissue did not show this difference (P = 0.126). During chemoradiation, the Topo I expression in tumour tissue of responders decreased significantly. TS did not show any differences between responders and nonresponders before treatment, but a significant decrease in the tumour tissue of responders was noted at the end of the treatment. Our data suggest that Topo I expression in rectal tumour mucosa might serve as a predictor of response to the neoadjuvant irinotecan-based chemoradiation, and hence might be a factor contributing to the development of individualized treatment.

Reduced expression of DNA topoisomerase I in SF295 human glioblastoma cells selected for resistance to homocamptothecin and diflomotecan.

Homocamptothecins (hCPTs) are a novel class of topoisomerase I (Top1) inhibitors with enhanced chemical stability compared with the currently used camptothecin (CPT) analogs irinotecan and topotecan. The hCPT derivative diflomotecan (BN80915) is currently in clinical trials. We established two resistant human glioblastoma cell lines, SF295/hCPT50 and SF295/BN50, by stepwise exposure of the parental SF295 line to increasing concentrations of hCPT and BN80915, respectively. The two resistant cell lines were 15- to 22-fold resistant to hCPT and BN80915 as well as 7- to 27-fold cross-resistant to other Top1 inhibitors, including CPT, topotecan, and the indenoisoquinolines MJ-III-65 (NSC 706744) and NSC 724998, but sensitive to the topoisomerase II inhibitors mitoxantrone and etoposide. Neither of the resistant cell lines displayed any detectable expression of the three major drug transporters P-glycoprotien, multidrug resistance-associated protein 1, or ATP-binding cassette, subfamily G (WHITE), member 2, as assessed by immunoblot or flow cytometry. Reduced expression of Top1 protein occurred at the transcriptional level in both of the resistant cell lines, consistent with the reduction of Top1 enzyme level as the major contribution to the resistance phenotype in SF295/hCPT50 and SF295/BN50 cells. Treatment of the resistant cell lines with the histone deacetylase inhibitor depsipeptide or the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine alone or concomitantly did not result in re-expression of Top1. Our studies suggest that selection for resistance to hCPT or BN80915 is primarily related to reduced Top1 expression at the transcriptional level, resulting in reduced enzyme levels.

Phase II study of sequentially administered low-dose mitomycin-C (MMC) and irinotecan (CPT-11) in women with metastatic breast cancer (MBC).

BACKGROUND: Preclinical studies show that mitomycin-C (MMC) followed by irinotecan (CPT-11) is synergistic. Therefore, we evaluated the toxicity and efficacy of sequentially administered low-dose MMC and CPT-11 in patients (pts) with pretreated metastatic breast cancer (MBC). Secondary objective was to evaluate the correlation between MMC-induced topoisomerase I (TOPO I) expression and NAD(P)H:quinone oxireductase 1 (NQO1) genotypes in peripheral blood mononuclear cells (PBMC) and efficacy or toxicity of the regimen. DESIGN: Thirty-two pts received MMC i.v. 6 mg/m(2) day 1 and CPT-11 i.v. 125 mg/m(2) days 2 and 8 every 28 days for maximum of six cycles. TOPO I expression and NQO1 reductase genotyping in 23 of 32 (72%) pts were assayed by PCR. RESULTS: The median time to progression (TTP) was 4.7 months (95% confidence interval 4.0-5.4 months). TOPO I expression was increased 5- to 10-fold and 20- to 30-fold in PBMC at 24 and 168 h, respectively. There was no relationship between these markers and efficacy or toxicity of the regimen. CONCLUSIONS: Sequential low-dose MMC and CPT-11 was active and tolerable by pretreated MBC pts. Future trials should focus on less pretreated MBC pts and sequential tumor biopsies to test the hypothesis that increased intratumoral expression of TOPO I is related to efficacy.

The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae.

RecQ DNA helicases and Topo III topoisomerases have conserved genetic, physical, and functional interactions that are consistent with a model in which RecQ creates a recombination-dependent substrate that is resolved by Topo III. The phenotype associated with Topo III loss suggests that accumulation of a RecQ-created substrate is detrimental. In yeast, mutation of the TOP3 gene encoding Topo III causes pleiotropic defects that are suppressed by deletion of the RecQ homolog Sgs1. We searched for gene dosage suppressors of top3 and identified Pif1, a DNA helicase that acts with polarity opposite to that of Sgs1. Pif1 overexpression suppresses multiple top3 defects, but exacerbates sgs1 and sgs1 top3 defects. Furthermore, Pif1 helicase activity is essential in the absence of Top3 in an Sgs1-dependent manner. These data clearly demonstrate that Pif1 helicase activity is required to counteract Sgs1 helicase activity that has become uncoupled from Top3. Pif1 genetic interactions with the Sgs1-Top3 pathway are dependent upon homologous recombination. We also find that Pif1 is recruited to DNA repair foci and that the frequency of these foci is significantly increased in top3 mutants. Our results support a model in which Pif1 has a direct role in the prevention or repair of Sgs1-induced DNA damage that accumulates in top3 mutants.

The human topoisomerase I gene promoter is regulated by NF-IL6.

We investigated the expression of the human DNA topoisomerase I (hTOP1) gene in HeLa cells and in adenovirus-transformed 293 cells. A highly conserved proximal promoter element is essential for hTOP1 promoter activity in HeLa cells but not in 293 cells. This correlates with the presence of specific promoter-binding proteins in HeLa cells and their absence in 293 cells. We identified the HeLa binding protein by screening a cDNA expression library with the specific promoter site as a probe and demonstrate now that the activating protein is identical to the nuclear factor for interleukin-6 expression (NF-IL6), a member of the C/EBP family of transcription factors. Overexpression of NF-IL6 strongly stimulates hTOP1 promoter activity in HeLa cells, suggesting that NF-IL6 is a major hTOP1-regulating protein. Because of the presence of adenovirus protein E1A, 293 cells express the hTOP1 gene more efficiently than HeLa cells but do not contain NF-IL6 activity. E1A activation of the hTOP1 promoter is suppressed by NF-IL6 overexpression. This result supports previous observations concerning a functional interaction between viral protein E1A and NF-IL6. Finally, we show that hTOP1 gene expression in differentiating macrophages is correlated with the synthesis of NF-IL6-specific mRNA.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Sensitivity to CPT-11 and platinum derivatives of stage III/Dukes' C colorectal cancer with occult neoplastic cells in lymph node sinuses.

The sensitivity of LN metastases to anticancer drugs such as CPT-11 and platinum agents was investigated by assessing Topo-1 and Bax/ERCC-1 expression in patients who had stage III/Dukes' C colorectal cancer with ONCs. In the recurrence group (RG) (n=21), immunohistochemical expression of Topo-1 was high in 8 patients (38.1%), and low in 13 patients (61.9%), while the non-recurrence group (N-RG) (n=12) showed high expression in 1 patient (8.3%) and low expression in 11 patients (91.7%) (not significant; N.S.). Regarding the immunohistochemical expression of Bax/ERCC-1, high Bax/low ERCC-1 expression was observed in 6 patients (28.6%) from the RG and other patterns of expression were seen in 15 patients (71.4%), while high Bax/low ERCC-1 expression level was observed in 3 patients (25.0%) from the N-RG and other patterns were found in 9 patients (75.0%) (N.S.). PCR analysis of Topo-1 expression in the RG (n=13) revealed high expression in 10 patients (76.9%) and low expression in 3 patients (23.1%), while the N-RG (n=3) showed high expression in 3 patients (100%) and low expression in none (N.S.). With respect to ERCC-1, PCR analysis of the RG (n=13) revealed high expression in 6 patients (46.2%) and low expression in 7 patients (53.8%), while the N-RG (n=3) showed high expression in 2 patients (66.7%) and low expression in 1 patient (33.3%) (N.S.). These results suggest that tumor sensitivity to CPT-11 and platinum derivatives is similar in stage III colorectal cancer patients with ONCs.

Sensitivity to CPT-11 and platinum derivatives of stage II/Dukes' B colorectal cancer with occult neoplastic cells in lymph node sinuses.

Among 13 patients with recurrent colorectal cancer (recurrence group: RG), immunohistochemical expression of Topo-1 was high in 4 patients (30.8%) and low in 9 patients (69.2%), while the non-recurrence group (N-RG) (n=8) showed high expression in 1 patient (12.5%) and low expression in 7 patients (87.5%) (NS). Regarding immunohistochemical expression of Bax/ERCC-1, high Bax/low ERCC-1 expression was observed in 6 patients (46.2%) from the RG and other patterns of expression were seen in 7 patients (53.8%), while high Bax/low ERCC-1 expression was observed in 4 patients (50.0%) from the N-RG and other patterns were found in 4 patients (50.0%) (NS). PCR analysis of Topo-1 expression revealed high expression in 9 patients (75.0%) from the RG (n=12) and low expression in 3 patients (25.0%), while the N-RG (n=8) showed high expression in all 8 patients (100.0%) and low expression in none (NS). With respect to ERCC-1, PCR analysis revealed high expression in 7 patients (58.3%) from the RG (n=12) and low expression in 5 patients (41.7%), while the N-RG (n=8) showed high expression in 1 patient (12.5%) and low expression in 7 patients (87.5%) (p<0.05). These results suggest that tumor sensitivity to CPT-11 and platinum derivatives is similar in stage II colorectal cancer patients with ONCs.

Expression of a molecular marker panel as a prognostic tool in gastric cancer patients treated postoperatively with docetaxel and irinotecan. A study of the Hellenic Cooperative Oncology Group.

INTRODUCTION: This study evaluated the prognostic role of vascular epidermal growth factor (VEGF), thymidylate synthase (TS), topoisomerase I (Topo-I), topoisomerase IIalpha (Topo-IIalpha) and E-cadherin (E-cadh) tumor expression, in patients with resectable gastric cancer, who were treated postoperatively with the docetaxel/irinotecan combination. PATIENTS AND METHODS: Forty-five patients with resectable gastric cancer were treated with 6 cycles of docetaxel 30 mg/m2 and irinotecan 110 m/m2 on day 1 and d8 every 21 days. All specimens were examined by using immunohistochemistry (IHC) for the expression of VEGF, TS, Topo-I, Topo-IIalpha and E-cadh. RESULTS: Positivity for TS was significantly correlated with age and for VEGF with diffuse histological type and good PS. No significant correlation was observed among Topo-I, Topo-IIalpha and E-cadh positivity with any of the clinicopathological parameters studied. Median overall survival (OS) was 31.7, and disease-free survival (DFS) 26 months, respectively. None of the above-investigated molecular markers were significantly associated with OS and DFS. Finally, according to the univariate analysis for survival, only advanced stages (III, IV) of the disease implied risk of death, mainly due to lymph node involvement and, to a lesser extent, tumor size. None of the studied molecular markers were found to be independent prognostic markers. CONCLUSION: These results should be interpreted very cautiously, due to the limited number of patients studied, as well as the limitations of the IHC technique.

Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes.

Topoisomerase I (Top1) is a ubiquitous enzyme that removes DNA supercoiling generated during transcription and replication. Top1 can be trapped on DNA as cleavage complexes by the anticancer drugs referred to as Top1 inhibitors as well as by alterations of the DNA structure. We reported recently that Top1 cleavage complexes (Top1cc) are trapped during apoptosis induced by arsenic trioxide and staurosporine. In the present study, we generalize the occurrence of apoptotic Top1cc in response to anticancer drugs, which by themselves do not directly interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form in the early phase of apoptosis and persist throughout the apoptotic process. Their formation is prevented by the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone and the antioxidant N-acetyl-L-cysteine. We propose that the trapping of Top1cc is a general process of programmed cell death, which is caused by alterations of the DNA structure (oxidized bases and strand breaks) induced by caspases and reactive oxygen species.

p53 dependence of topoisomerase I recruitment in vivo.

DNA damage is attended by rapid recruitment of endogenous type I topoisomerase (topo I) into covalent cleavage complexes with genomic DNA in vivo. In contrast, endogenous topoisomerase II alpha and beta are not stimulated by DNA damage. We show that topo I and p53 are able to associate at arrested topo I-genomic DNA covalent complexes in vivo, suggesting that p53 directly stimulates topo I activity and damage to the genome of the afflicted cell. Moreover, cells that express wild-type p53 are most proficient at recruiting topo I after DNA damage; however, the p53 dependence is conditional because topo I recruitment after DNA damage can be restored if p53 mutant cells (containing a single mutant allele) are artificially held in G1. In contrast, p53 null mutants do not recruit topo I after DNA damage under any conditions (although camptothecin-dependent topo I/DNA complexes readily form in the nulls). These results show that topo I activation after DNA damage depends on the p53 status of the cell. It also depends upon the cell cycle in a way that is very different from that observed with DNA replication-dependent, camptothecin-mediated DNA breaks. The data suggest a model where p53 activates topo I, which inflicts additional genomic damage after the initial UV damage events. Topoisomerases therefore contribute to the p53 commitment to apoptosis, and topo I might assist in elimination of DNA-damaged cells as part of the cellular proofreading function inherent in the p53 pathway.

The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorectal cancer cells.

The DNA mismatch repair (MMR) system is involved in the correction of base/base mismatches and insertion/deletion loops arising during replication. In addition, some of the MMR components participate in recombination and double-strand break repair as well as cell cycle regulation and apoptosis. The inactivation of MMR genes, usually hMSH2 or hMLH1, is associated with human colorectal cancers and is responsible for the characteristic microsatellite instability (MSI)+ phenotype of these tumors. Because MMR is assumed to modulate cytotoxicity to various chemotherapeutic agents that act upon DNA, our objectives have been to define its possible involvement in the cytotoxicity of topoisomerase inhibitors. We have shown that colorectal cancer cell lines defective in DNA MMR exhibit an increased sensitivity to both camptothecin, a topoisomerase I inhibitor, and etoposide, a topoisomerase II inhibitor. Sensitivity to these drugs cannot be predicted by measuring endogenous levels of topoisomerase I and II. Our results also indicate that neither p53 status, nor cell cycle alterations correlate with the sensitivity of colorectal cancer cells to topoisomerase inhibitors. On the other hand, our data showing that resistance to these drugs can be achieved by the functional complementation of hMLH1 in an hMLH1-defective cell line have allowed us to establish that MMR is a critical determinant for chemosensitivity. Interestingly, our observations provide the rationale for the better responsiveness of MSI+ tumors to CPT-11, a camptothecin derivative, which we have observed in patients with metastatic colorectal cancers.

Drug-induced down-regulation of topoisomerase I in human epidermoid cancer cells resistant to saintopin and camptothecins.

The anticancer agent saintopin induces DNA cleavage mediated by both topoisomerase (topo) I and topo II in vitro through stabilization of the reversible enzyme-DNA cleavable complex. We established saintopin-resistant cell lines (KB/STP-1 and KB/STP-2) from human epidermoid cancer KB cells by stepwise exposure to increasing doses of the drug. KB/STP-1 and KB/STP-2 cells showed 12- and 44-fold increases, respectively, in resistance to saintopin relative to that of KB cells. Both saintopin-resistant cell lines showed only small reductions in sensitivity to the topo II inhibitor etoposide but developed marked cross-resistance to the topo I-targeting camptothecin derivative CPT-11 [(4s)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbony loxy] dione hydrochloride trihydrate] and its active form, SN-38 (7-ethyl-10-hydroxycamptothecin). In contrast, both KB/STP-1 and KB/STP-2 cells showed increased collateral sensitivity to cisplatin, a nitrosourea derivative, mitomycin C, and UV light. The protein concentration, activity, and mRNA abundance of both topo I and topo II were similar in KB/STP-1, KB/STP-2, and the parental KB cells. There were no significant changes in the drug-stabilized topo-DNA cleavable complex formation in KB and KB/STP-2 cells. Two point mutations were detected in topo I cDNA from KB/STP-2 cells, but these were also present in KB cells. Topo I mRNA abundance decreased markedly immediately after exposure of KB/STP-2 cells to saintopin; no such effects were apparent in KB cells. In contrast, topo II mRNA was not markedly affected by saintopin in either KB or KB/STP-2 cells. Treatment with CPT-11 or SN-38 also induced a markedly greater and more persistent reduction in topo I mRNA abundance in KB/STP-2 cells than in KB cells. Etoposide had no marked effect on topo I mRNA abundance in either KB/STP-2 or KB cells. Topo I mRNA was highly unstable in KB/STP-2 cells in comparison to KB cells when incubated with saintopin. This novel regulation of topo I mRNA by topo I-targeting agents could be associated with acquirement of drug resistance to saintopin or SN-38/CPT-11 in KB/STP-2 cells.