Artificial Intelligence Systems as Prognostic and Predictive Tools in Ovarian Cancer.

BACKGROUND: The ability to provide accurate prognostic and predictive information to patients is becoming increasingly important as clinicians enter an era of personalized medicine. For a disease as heterogeneous as epithelial ovarian cancer, conventional algorithms become too complex for routine clinical use. This study therefore investigated the potential for an artificial intelligence model to provide this information and compared it with conventional statistical approaches. METHODS: The authors created a database comprising 668 cases of epithelial ovarian cancer during a 10-year period and collected data routinely available in a clinical environment. They also collected survival data for all the patients, then constructed an artificial intelligence model capable of comparing a variety of algorithms and classifiers alongside conventional statistical approaches such as logistic regression. RESULTS: The model was used to predict overall survival and demonstrated that an artificial neural network (ANN) algorithm was capable of predicting survival with high accuracy (93 %) and an area under the curve (AUC) of 0.74 and that this outperformed logistic regression. The model also was used to predict the outcome of surgery and again showed that ANN could predict outcome (complete/optimal cytoreduction vs. suboptimal cytoreduction) with 77 % accuracy and an AUC of 0.73. CONCLUSIONS: These data are encouraging and demonstrate that artificial intelligence systems may have a role in providing prognostic and predictive data for patients. The performance of these systems likely will improve with increasing data set size, and this needs further investigation.

Characterisations of human prostate stem cells reveal deficiency in class I UGT enzymes as a novel mechanism for castration-resistant prostate cancer.

BACKGROUND: Evidence increasingly supports that prostate cancer is initiated by the malignant transformation of stem cells (SCs). Furthermore, many SC-signalling pathways are shown to be shared in prostate cancer. Therefore, we planned transcriptome characterisation of adult prostate SCs as a strategy to consider new targets for cancer treatment. METHODS: Intuitive pathway analysis was used for putative target discovery in 12 matched selections of human prostate SCs, transiently amplifying cells and terminally differentiated cells. These were pooled into three groups according to the stage of differentiation for mRNA microarray analysis. Targets identified were validated using uncultured primary tissue (n=12), functional models of prostate cancer and a tissue microarray consisting of benign (n=42) and malignant prostate (n=223). RESULTS: A deficiency in class 1 UDP glucuronosyltransferase (UGT) enzymes (UGT1A) was identified in prostate SCs, which are involved in androgen catabolism. Class 1 UGT enzyme expression was also downregulated in cancer SCs and during progression to metastatic castration-resistant prostate cancer (CRPC). Reduction of UGT1A expression in vitro was seen to improve cell survival and increase androgen receptor (AR) activity, as shown by upregulation of prostate-specific antigen expression. INTERPRETATION: Inactivation of intracellular androgen catabolism represents a novel mechanism to maintain AR activity during CRPC.

Upregulated FGFR1 expression is associated with the transition of hormone-naive to castrate-resistant prostate cancer.

BACKGROUND: Prostate cancer (PC) represents a global health issue. Treatment for locally advanced and metastatic PC remains unsatisfactory. The androgen receptor (AR) has been validated in having a key role in both naïve and castrate-resistant PC (CRPC). However, the significance of other signalling pathways in CRPC is less well validated. METHODS: To gain a better insight into the molecular signalling cascades involved in clinical CRPC, we performed gene expression profiling using the Illumina DASL assay and studied matched hormone-naive (HN) and CR prostate tumours (n=10 pairs). Ingenuity Pathways Analysis (IPA) was used to identify potential networks involved, and further validation was performed in in vitro cell models and clinical tumours. RESULTS: Expression of 50 genes was significantly different between HN and CRPC. IPA revealed two networks of particular interest, including AR and FGFR1, respectively. FGFR1 expression was confirmed to be significantly upregulated in CRPC (P ≤ 0.005), and abnormal FGFR1 expression was associated with shorter time to biochemical relapse in HNPC (P=0.006) and less favourable disease-specific survival in CRPC (P=0.018). CONCLUSION: For the first time, our gene expression profiling experiment on archival tumour materials has identified upregulated FGFR1 expression to be associated with PC progression to the CR state.

Pre-clinical evaluation of cyclin-dependent kinase 2 and 1 inhibition in anti-estrogen-sensitive and resistant breast cancer cells.

BACKGROUND: Cellular proliferation, driven by cyclin-dependent kinases (CDKs) and their cyclin partners, is deregulated in cancer. Anti-estrogens, such as tamoxifen, antagonise estrogen-induced ERalpha transactivation of cyclin D1, resulting in reduced CDK4/6 activity, p27(Kip1)-mediated inhibition of CDK2 and growth arrest. We hypothesised that direct inhibition of CDK2 and CDK1 may overcome the major clinical problem of anti-estrogen resistance. METHODS: The cellular effects of CDK2/1 siRNA knockdown and purine-based CDK2/1 inhibitors, NU2058 and NU6102, were measured in anti-estrogen-sensitive and resistant breast cancer cell lines. RESULTS: CDK2 knockdown caused G1 accumulation, whereas CDK1 depletion caused G2/M slowing, and dual CDK1/2 depletion resulted in further G2/M accumulation and cell death in both anti-estrogen-sensitive and resistant cells, confirming CDK2 and CDK1 as targets for breast cancer therapy. In contrast to tamoxifen, which only affected hormone-sensitive cells, NU2058 and NU6102 reduced CDK2-mediated phosphorylation of pRb, E2F transcriptional activity and proliferation, ultimately resulting in cell death, in both anti-estrogen-sensitive and resistant cells. Both drugs caused G2/M arrest, reflective of combined CDK2/1 knockdown, with a variable degree of G1 accumulation. CONCLUSION: These studies confirm the therapeutic potential of CDK2 and CDK1 inhibitors for cancer therapy, and support their use as an alternative treatment for endocrine-resistant breast cancer.

Similar expression to FGF (Sef) inhibits fibroblast growth factor-induced tumourigenic behaviour in prostate cancer cells and is downregulated in aggressive clinical disease.

BACKGROUND: The fibroblast growth factor (FGF) axis is an important mitogenic stimulus in prostate carcinogenesis. We have previously reported that transcript level of human similar expression to FGF (hSef), a key regulator of this pathway, is downregulated in clinical prostate cancer. In this study we further analysed the role of hSef in prostate cancer. METHODS: hSef function was studied in in vitro and in vivo prostate cancer models using stable over-expression clones. Protein expression of hSef was studied in a comprehensive tissue microarray. RESULTS: Stable over-expression of hSef resulted in reduced in vitro cancer cell proliferation, migration and invasive potential. In an in vivo xenograft model, the expression of hSef significantly retarded prostate tumour growth as compared with empty vector (P=0.03) and non-transfected (P=0.0001) controls. Histological examination further showed a less invasive tumour phenotype and reduced numbers of proliferating cells (P=0.0002). In signalling studies, hSef inhibited FGF-induced ERK phosphorylation, migration to the nucleus and activation of a reporter gene. Constitutively active Ras, however, was able to reverse these effects, suggesting that hSef exerts an effect either above or at the level of Ras in prostate cancer cells. In a large tissue microarray, we observed a significant loss of hSef protein in high-grade (P<0.0001) and metastatic (P<0.0001) prostate cancer. CONCLUSIONS: Considered together, the role of hSef in attenuating FGF signalling and evidence of downregulation in advanced tumours argue strongly for a tumour suppressor function in human prostate cancer.

Androgen-related expression of G-proteins in ovarian cancer.

BACKGROUND: Epidemiological and in vitro data implicate androgens in the aetiology of ovarian cancer, but the mechanisms by which this is mediated are unclear. In this study, we wished to examine the effects of androgens on gene expression in ovarian cancer. METHODS: The expression of androgen receptor (AR) in OVCAR3 and OSEC2 cells was confirmed using immunoblotting and response to androgens was measured using flow cytometric assessment of S-phase fraction. The differential gene expression between androgen stimulated and unstimulated OVCAR3 ovarian cancer cells was examined with a cDNA microarray. The upregulation of a subset of these genes was then confirmed with reverse transcriptase PCR in both OVCAR3 and OSEC2, an ovarian epithelial cell line. Finally, the clinical significance of this upregulation was investigated by examining the expression of Rab25 and Rab35, two G-protein-related molecules in an ovarian cancer tissue microarray (TMA). RESULTS: OVCAR3 and OSEC2 cells were shown to express the AR and showed an increase in S-phase fraction in response to androgen treatment. Treatment of OVCAR3 cells with androgen resulted in a significant upregulation of 121 genes. These findings were confirmed for a subset of seven monomeric G-protein-related genes in both OVCAR3 and OSEC2 cells. After staining for Rab25 and Rab35, the majority of TMA sections examined showed expression for Rab25 (92%) and Rab35 (95%). The expression of Rab25 correlated with histological grade, and expression was higher in endometrioid (median histoscore 10.5) than serous (7.5) or mucinous (5.3) tumours. The expression of Rab25 correlated positively with AR expression supporting its role as an androgen responsive gene in ovarian cancer. CONCLUSIONS: These results suggest that androgens can effect expression of the oncogenic GTPases in ovarian cancer. We propose that the androgen responsive Rab35 may have clinical importance as a biomarker of AR function.

Correction: The induction of core pluripotency master regulators in cancers defines poor clinical outcomes and treatment resistance.

The original version of this article contained an error in Fig. 5a where the colours of the labels representing the Hinge and LBD of the AR were incorrect and did not match the corresponding exons. The corrected version of this Figure now appears in the article. The conclusions of this paper were not affected. The authors apologise for this error and any confusion caused.

The induction of core pluripotency master regulators in cancers defines poor clinical outcomes and treatment resistance.

Stem cell characteristics have been associated with treatment resistance and poor prognosis across many cancer types. The ability to induce and regulate the pathways that sustain these characteristic hallmarks of lethal cancers in a novel in vitro model would greatly enhance our understanding of cancer progression and treatment resistance. In this work, we present such a model, based simply on applying standard pluripotency/embryonic stem cell media alone. Core pluripotency stem cell master regulators (OCT4, SOX2 and NANOG) along with epithelial-mesenchymal transition (EMT) markers (Snail, Slug, vimentin and N-cadherin) were induced in human prostate, breast, lung, bladder, colorectal, and renal cancer cells. RNA sequencing revealed pathways activated by pluripotency inducing culture that were shared across all cancers examined. These pathways highlight a potential core mechanism of treatment resistance. With a focus on prostate cancer, the culture-based induction of core pluripotent stem cell regulators was shown to promote survival in castrate conditions-mimicking first line treatment resistance with hormonal therapies. This acquired phenotype was shown to be mediated through the upregulation of iodothyronine deiodinase DIO2, a critical modulator of the thyroid hormone signalling pathway. Subsequent inhibition of DIO2 was shown to supress expression of prostate specific antigen, the cardinal clinical biomarker of prostate cancer progression and highlighted a novel target for clinical translation in this otherwise fatal disease. This study identifies a new and widely accessible simple preclinical model to recreate and explore underpinning pathways of lethal disease and treatment resistance.

Loss of Sef (similar expression to FGF) expression is associated with high grade and metastatic prostate cancer.

Fibroblast growth factors (FGF), and in particular FGF8, have been strongly implicated in prostate carcinogenesis. This study investigated the expression of Sef, a key inhibitory regulator of FGF signalling, in prostate cancer. In a panel of cell lines, hSef was detected in both androgen-dependent and independent cells but was significantly reduced in highly metastatic derivative clones. hSef expression was not influenced by androgenic stimulation. Forced downregulation of hSef by siRNA increased FGF8b induced cell migration (P=0.02) and invasion (P=0.007). Reduced hSef levels also enhanced FGF8b stimulated expression of MMP9 (P=0.005). mRNA in situ hybridization revealed hSef expression in 80% (8/10) of benign biopsies but in only 69% (23/33) of Gleason sum 4-7 and 35% (10/28) of Gleason sum 8-10 cancer biopsies (P=0.004). Quantitative PCR of microdissected glands confirmed this trend (P=0.001). hSef was expressed in 69% (27/39) of non-metastatic tumours but in only 18% (2/11) of metastatic tumours (P=0.004, n=50). hSef expression was next correlated with earlier data on FGF8b expression in a subgroup of cancers. In this cohort, 86% (19/22) of high-grade cancers expressed FGF8 but only 31% (7/22) expressed hSef. Positive FGF8 expression but a loss of hSef was observed in 88% (7/8) of metastatic tumours. In contrast, metastasis was evident in only 10% (1/10) of tumours, which co-expressed both FGF8 and hSef (P<0.001). These results suggest evidence that hSef is downregulated in advanced prostate cancer and might facilitate an enhanced tumorigenic response to FGFs. Further research into the role of hSef in cancer cell signalling and the mechanism of its downregulation may contribute to more effective targeting of growth factors in prostate cancer.

Expression of GnRH type II is regulated by the androgen receptor in prostate cancer.

GnRH II has important functional effects in steroid hormone-dependent tumours. Here we investigated the expression and regulation of GnRH II in prostate cancer. GnRH II protein was equally expressed in benign (73%) and malignant (78%) biopsies studied in a prostate tissue microarray (P = 0.779). There was no relationship between expression and clinical parameters in the cancer cohort. GnRH II was, however, significantly reduced in tumour biopsies following hormone ablation. This was further investigated in a prostate xenograft model where androgens increased GnRH II levels, while their withdrawal reduced it. In cell lines, we confirmed high levels of GnRH II in androgen receptor (AR)-positive LNCaP cells but low levels in AR-negative PC3 cells. In LNCaP cells, GnRH II induction by androgens was blocked by the AR inhibitor casodex, but not by cycloheximide treatment. Sequence analysis subsequently revealed a putative androgen response element in the upstream region of the GnRH II gene and direct interaction with the AR was confirmed in chromatin immunoprecipitation experiments. Finally, to test whether the effects of GnRH II were dependent on AR expression, LNCaP and PC3 cells were exposed to exogenous peptide. In both cell lines, GnRH II inhibited cell proliferation and migration, suggesting that its function is independent of AR status. These results provide evidence that GnRH II is widely expressed in prostate cancer and is an AR-regulated gene. Further studies are warranted to characterise the effects of GnRH II on prostate cancer cells and investigate its potential value as a novel therapy.

Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding.

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

Reversal of multidrug resistance by verapamil and modulation by alpha 1-acid glycoprotein in wild-type and multidrug-resistant Chinese hamster ovary cell lines.

Multidrug resistance can be reversed by a range of "calcium channel"-blocking drugs in vitro, of which verapamil is the most widely used. Verapamil is bound to alpha 1-acid glycoprotein (AAG) in vivo in humans but is absent from calf serum, used in tissue culture media. The effect of AAG on the ability of verapamil to alter Adriamycin cytotoxicity was assessed in parental Chinese hamster ovary cells (CHO-K1) and in a multidrug-resistant subline (CHO-Adrr). In both the parental and the resistant cells, there was a dose-related potentiation of Adriamycin cytotoxicity by verapamil. At 10 microM verapamil, there was a 5-fold decrease in the concentration of Adriamycin that caused 50% reduction in growth of CHO-K1 cells but a 15-fold decrease in CHO-Adrr cells. In the presence of increasing AAG concentrations within the range found in cancer patients, there was a concentration-related reduction in the effects of verapamil. In CHO-Adrr cells, there was complete reversal of the potentiating effect of 10 microM verapamil at 2 mg/ml AAG. In contrast, in CHO-K1 cells, AAG reduced the effects of verapamil by only 20% at a similar concentration. There was a much higher internal uptake of fluorescein-labeled AAG by CHO-Adrr cells than by CHO-K1 cells. These results suggest that, in addition to a plasma membrane site, there may be a major endosomal site of action of verapamil in multidrug-resistant cells. The implications are that verapamil in vivo in the presence of AAG may effectively reverse low levels of multidrug resistance, but not high levels. Thus selection of patients with low AAG levels may be appropriate for clinical studies.

Isolation and characterization of Chinese hamster ovary cell lines sensitive to mitomycin C and bleomycin.

Seven Chinese hamster ovary K1 cell lines exhibiting sensitivity to anticancer drugs have been isolated by a replica-plating technique. Five of the mutants are hypersensitive (approximately 7-fold, as judged by D37 values) to the DNA cross-linking agent mitomycin C. Of these, one is also appreciably sensitive to UV light. Significant variations in their cross-sensitivity to cis-platinum(II) diammine dichloride, chlorambucil, and Adriamycin have also been observed. Two additional mutants have been isolated on the basis of sensitivity to the radiomimetic agent bleomycin. One of these shows greater than 6-fold sensitivity to bleomycin, while the other is approximately 14 times more sensitive than the parental strain to bleomycin and is also hypersensitive to a number of other DNA-damaging agents, including cis-platinum(II) diammine dichloride, chlorambucil, X-rays, and UV light. Both bleomycin-sensitive mutants also exhibit some degree of sensitivity to Adriamycin. In all cases, the cell lines have been grown in continuous culture for 3 months without evidence of reversion and should act as suitable recipients in DNA transfection experiments aimed at identifying human DNA repair genes.

Cross-sensitivity to topoisomerase II inhibitors in cytotoxic drug-hypersensitive Chinese hamster ovary cell lines.

We have isolated a Chinese hamster ovary cell line, designated ADR-1, which exhibits hypersensitivity to a range of drugs which are thought to inhibit the action of the enzyme topoisomerase II. These include anthracyclines, other classes of intercalating agents, and the epipodophyllotoxin, etoposide. No significant sensitivity to radiation, or to mono- and bifunctional alkylating agents was seen, although mild cross-sensitivity to the radiomimetic agent bleomycin was observed. We have monitored the level of DNA strand breaks induced by topoisomerase II inhibitors in ADR-1 cells using alkaline elution. At equimolar Adriamycin (doxorubicin) doses, more protein-associated DNA strand breaks are induced in ADR-1 cells than in wild-type cells. This enhanced level of drug-induced strand breaks does not appear to be a function of increased drug uptake as both lines accumulate similar levels of radiolabeled daunomycin. Both the rate of repair of strand breaks and the final percentage of strand breaks rejoined was equivalent in the 2 cell lines. These results are consistent with an enhancement in the level of topoisomerase II-dependent DNA breakage in ADR-1 cells following exposure to topoisomerase II inhibitors. We have previously reported the isolation of 2 bleomycin-sensitive Chinese hamster ovary cell lines, BLM-1 and BLM-2 (C. N. Robson et al., Cancer Res. 45:5304-5309, 1985). While BLM-1 exhibited cross-sensitivity only to Adriamycin, BLM-2 was shown to be hypersensitive not only to Adriamycin out also to certain alkylating agents and to ionizing radiation. In this paper, we show that both BLM-1 and BLM-2 also exhibit mild cross-sensitivity to a range of topoisomerase II inhibitors. These results indicate that intercalating agents and epipodophyllotoxins exert their cytotoxicity via common mechanisms and suggest that the maintenance of normal levels of cellular resistance to these agents requires the products of several different genes.

Isolation and characterization of a Chinese hamster ovary cell line resistant to bifunctional nitrogen mustards.

A drug-resistant derivative of a Chinese hamster ovary cell line has been generated by chronic exposure to progressively higher concentrations of chlorambucil. The cells exhibit greater than 20-fold resistance to the cytotoxic effects of chlorambucil and comparable levels of cross-resistance to mechlorethamine and melphalan. These drugs all belong to a class of bifunctional alkylating agents which generate DNA cross-links by reaction at the N-7 position of guanine. However, no resistance is observed to several other drugs which possess a similar mechanism of action, to cis-platinum diammine dichloride or to bischloroethylnitrosourea and mitomycin C, which cross-link DNA via the O6 position of guanine. Lack of resistance to vincristine, colchicine, or Adriamycin coupled with the failure of the calcium channel blocker verapamil to reverse the phenotype, indicates that the mechanism of resistance is distinct from that characterized by the multidrug-resistant phenotype. Support for this view comes from the finding that no significant alteration in melphalan uptake could be demonstrated. The phenotype is very stable and has been maintained during 12 months of continuous culture without further selection. A slightly elevated basal level of glutathione is present in the resistant cells, but resistance is not overcome by depletion of intracellular glutathione with buthionine sulfoximine. A cytosolic protein with a molecular weight of approximately 25,000 is constitutively overexpressed in the resistant cells. Although these cells have an abnormal karyotype, no conclusive evidence for any cytogenetic indicator of gene amplification could be detected.

Possible role of inhibition of glutathione S-transferase in the partial reversal of chlorambucil resistance by indomethacin in a Chinese hamster ovary cell line.

We have reported previously the isolation and characterization of a Chinese hamster ovary cell line, designated CHO-Chlr, which exhibits resistance to bifunctional nitrogen mustards while maintaining sensitivity to a range of other alkylating agents and chemotherapeutic drugs. This enhanced drug resistance is associated with a greater than 40-fold increase in the level of expression of an alpha class (YcYc) glutathione S-transferase (GST) as compared to the parental, CHO-K1, cell line. Here, we have purified GST from CHO-Chlr cells and show that the nonsteroidal antiinflammatory drug indomethacin acts as an inhibitor of enzyme activity. Indomethacin at 500 microM causes no significant decrease in colony forming ability of either CHO-K1 or CHO-Chlr cells. However, the cytotoxicity of chlorambucil is potentiated 5.5-fold in CHO-Chlr cells, but only 2.5-fold in CHO-K1 cells following preexposure to 500 microM indomethacin. In contrast, the antiinflammatory agent acetylsalicylic acid failed to inhibit the activity of purified GST and caused no potentiation of chlorambucil toxicity, suggesting that the potentiation by indomethacin is not due to the effects of this drug on prostaglandin synthesis. These studies provide further evidence that GSTs may be involved in the development of resistance to bifunctional alkylating agents and suggest that indomethacin, or agents with similar activities, may be of value as an adjunct to chemotherapy in some patients with tumors resistant to treatment with alkylating agents.

Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue.

Skeletal metastases are common in advanced prostate cancer, causing considerable morbidity, and they are usually osteoblastic in nature with no clear explanation for this phenomenon. Bone morphogenetic proteins (BMPs) induce bone formation in vivo, and preliminary work showed a possible association between BMPs and prostatic skeletal metastases; differential expression favors BMP-6 as a potential new marker and mediator of osteosclerotic deposit formation. We investigated BMP-6 mRNA and protein expression by in situ hybridization and immunohistochemistry in malignant and benign prostates from 40 men. BMP-6 mRNA expression was detected exclusively in malignant epithelial cells in 20 of 21 patients (95%) with metastases and in 2 of 11 patients (18%) with localized cancer, and it was absent in 8 benign samples. Immunostaining for BMP-6 was predominantly cytoplasmic and was present in all primary tumors with established metastases and in 4 of 11 (36%) organ-confined cancers. In benign prostatic hyperplasia, basal cells and areas of basal cell hyperplasia were positive for BMP-6 by immunohistochemistry. The results suggest a close association between BMP-6 expression in primary malignant prostatic tissue and skeletal metastases. BMP-6 may be responsible, in part, for the osteoblastic changes in metastatic lesions secondary to prostate cancer.

Reduced levels of drug-induced DNA cross-linking in nitrogen mustard-resistant Chinese hamster ovary cells expressing elevated glutathione S-transferase activity.

We have reported previously (C. N. Robson et al., Cancer Res., 46: 6290-6294, 1986) the isolation of a Chinese hamster ovary cell line, designated CHO-Chlr, that exhibits resistance to bifunctional nitrogen mustards while maintaining the normal parental level of sensitivity to several other alkylating agents. We have compared the rate of formation and repair of DNA cross-links induced by mechlorethamine in CHO-Chlr and parental CHO-K1 cells, both in intact cells and in isolated nuclei. Equimolar doses of mechlorethamine induce significantly fewer DNA interstrand cross-links in CHO-Chlr cells than in CHO-K1 cells, but levels of DNA-protein adducts are approximately equivalent in the two lines. There is a correlation between the relative resistance of CHO-Chlr cells to mechlorethamine (34-fold) and the amount of drug required to induce approximately equal numbers of DNA interstrand cross-links in the two cell lines. This strongly implicates DNA-DNA adducts in the cytotoxic action of mechlorethamine. DNA cross-linking studies on isolated nuclei reveal only minor differences between the two lines even with identical drug treatments. The rate of cross-link repair is comparable in the two cell lines. These results, taken together with our earlier observation that the rate of drug accumulation is identical in these two lines, suggest that enhanced cytoplasmic drug detoxification is the underlying resistance mechanism in CHO-Chlr cells. We have measured cellular glutathione S-transferase activity, using both the general substrate 1-chloro-2,4-dinitrobenzene, and substrates with some specificity for the different classes of transferase isoenzymes. Total enzyme activity (as measured with 1-chloro-2,4-dinitrobenzene) is elevated 3-fold in the resistant cells. A 2- and 5-fold increase, respectively, in activity against ethacrynic acid and cumene hydroperoxide is detectable in CHO-Chlr cells. This elevation in catalytic activity in the resistant cells is reflected in higher levels of both the Yf- and Ya-type transferase subunits.

Decreased NADPH:cytochrome P-450 reductase activity and impaired drug activation in a mammalian cell line resistant to mitomycin C under aerobic but not hypoxic conditions.

Mitomycin C (MMC) is regarded as the prototype bioreductive alkylating agent in clinical use. To elucidate the biochemical basis of MMC resistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure to progressively higher concentrations of MMC. CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C. In contrast, CHO-MMC cells showed only a 2-fold level of resistance to BMY 25282, a more easily activated analogue of MMC, and exhibited parental sensitivity to MMC under radiobiologically hypoxic conditions. CHO-MMC cells showed no increased resistance to a range of DNA damaging agents including several other alkylating agents (e.g., melphalan and methyl methanesulfonate). Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vincristine) was present only at very low levels. Using a specific high performance liquid chromatography technique, we examined the rates of reduction of MMC and BMY 25282 in cell extracts from CHO-K1 and CHO-MMC cells under both aerobic (air) and hypoxic (N2) conditions. Reduction rates for both drugs were at least 30-fold faster under nitrogen than in air. Metabolism of MMC was undetectable in air but was readily detectable under nitrogen and was 2- 3-fold slower in CHO-MMC cell extracts than in CHO-K1 cell extracts. Although BMY 25282 was more readily reduced under nitrogen, no difference was detected between extracts from CHO-K1 or CHO-MMC cells in the rate of reduction of BMY 25282 under either air or nitrogen. The activity of NADPH:cytochrome P-450 (cytochrome c) reductase, an enzyme implicated in the bioreductive activation of MMC, was 3-4-fold lower in CHO-MMC cells than in the parental line. These findings suggest that the resistance of CHO-MMC cells to MMC under aerobic conditions may be due to impaired metabolic activation of the drug as a result of a decrease in NADPH:cytochrome P-450 reductase activity. This supports the view that decreased bioreductive enzyme activity may be a significant mechanism for acquired resistance to MMC in tumor cells in vivo and that more readily activated analogues may be potentially useful in overcoming this specific form of resistance.