CANCER DRUG RESISTANCE

Our continuing research efforts focus on determining how tumor cells develop and express resistance to anticancer drugs. By defining those molecular events that lead to acquired drug resistance, we can understand the adaptive changes that enable cells the selective advantage of survival. These traits, once understood, can guide rational therapeutic approaches and can provide insight into the mechanism of action of novel anticancer drugs.

The peptidomimetic drug, g-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine diethyl ester, TER199, (Telik Inc.) is an analog of glutathione (GSH) designed to be an isozyme specific inhibitor of glutathione Stransferase (GST) P1-1. This compound, and the deesterified moiety TER117, is shown to be an effective inhibitor of multidrug resistance associated protein (MRP1)-mediated drug resistance. Kinetic analyses revealed that TER117 non-competitively inhibits the transport of 2,4-dinitrophenyl-S-glutathione, a prototypical MRP-1 ligand, with a Ki of 760 mM. TER199 reversed the accumulation deficit of daunorubicin in MRP1 transfected NIH3T3 fibroblasts and maintained intracellular levels of daunorubicin for greater than two hours after drug removal. Cytotoxicity assays revealed that TER199 significantly reversed the resistance of MRP1 transfected NIH3T3 cells for doxorubicin, etoposide, vincristine and mitoxantrone. HL60 cells made resistant to TER199 by chronic, long-term selection had increased mRNA and protein levels of MRP1 and gglutamyl cysteine synthetase (g-GCS) heavy and light subunits; g-GCS is the rate limiting enzyme in GSH synthesis. Increased mRNA levels for ATP-binding cassette transporter 2, ABC2, a novel member of the ABC superfamily, was also observed. In spite of increased gGCS, the glutathione content of TER199-resistant HL60 cells was reduced approximately 35% from that of parental HL60 cells. These cells also exhibited a drug resistance profile commensurate with the previously described MRP1 over-expressing phenotype, with resistance to vinca alkaloids, epipodophyllotoxins, and anthracyclines. Furthermore, additional cross resistance to taxol, mitoxantrone and 5fluorouracil was observed. These data extend the interpretation of the mechanism of action of the peptidomimetic to include the membrane-transporter, MRP, as a cellular target.

Resistance to anticancer drugs is often associated with overexpression of the detoxification isozyme GSTP1-1. Recently, a GSTP1-1 knock out (KO) mouse model was developed by Henderson, et al. (Proc. Natl. Acad. Sci. 95:5275, 1998). We have established mouse embryonic fibroblast (MEF) cell lines from WT-129 and GSTP1-1 KO mice to identify a possible phenotype linked with GSTP1-1 inactivation.

Absence of GSTP1-1 protein and DNA expression was determined in liver and MEF cells obtained from GSTP1-1 KO mice. While no differences were observed in cell morphology or volume between wild type (WT) and KO MEF, cell doubling time was faster in KO cells compared to WT cells (24 hours versus 36 hours, respectively). Differences in cell growth were maintained in the presence of the GSTP1-1 inhibitor, TER199. There was no difference in plating efficiency between WT and KO cells. Future experiments will investigate possible differences in cell cycle control. Additionally, we have characterized a subline of WT and KO MEF cells that appear to have immortalized. These cell lines maintain the null phenotype expression and cell doubling rate. These results suggest that, in addition to its known catalytic detoxification properties, GSTP1-1 may function in a ligand binding fashion and influence cell growth and proliferation.

TER199, a paralog of glutathione was developed as a specific GSTP1-1 inhibitor and modulator of drug resistance. Secondary to this, TER199 was found to elicit a proliferative response in bone marrow stem cells. This observation was investigated using colony forming assays conducted with bone marrow from 129 (WT) mice and GSTP11 KO mice (Henderson et al., Proc. Natl. Acad. Sci. 95:5275, 1998). Studies with MEF derived from WT and GSTP1-1 KO mice showed doubling times of 34.8±12.3 hours and 24.8 ± 8.4 hours respectively, further implicating GSTP1-1 in cell proliferation. Mechanistically, cell proliferation can be attributed to mild oxidative stress. In order to explore this with regard to TER199, bone marrow cells were assayed for production of the superoxide anion in the presence of TER199 (0-200 mM). While TER199 was not found to stimulate superoxide production itself, it was found to reduce the response of cells to stimulation by a phorbol ester. Thus, TER199 appears to be acting as an antioxidant. Ongoing work will evaluate further the mechanisms involved in TER199-induced bone marrow proliferation and the involvement of GSTP1-1 in proliferation.

TER286 [g-glutamyl-a-amino-b(2-ethyl-N,N,N',N'-tetrakis (2-chloroethyl) phos-phorodiamidate)-sulfonyl)-propionyl-(R)-(-) phenylglycine], a novel nitrogen mustard prodrug that is preferentially activated by GSTP1-1 is presently in preclinical development. An HL60/TER286 resistant cell line was selected by chronic, long-term exposure. While resistance was not readily achieved, eventually a 5-fold resistant clone was isolated [7.5 ± 2.8 versus 36.4 ± 5.3 mM]. Five-fold cross-resistance to melphalan occurred, but there were no differences in cytotoxicity to adriamycin, taxol and TER199. While RT-PCR results showed no significant changes in transcript for GST a, m, and p, both the catalytic and regulatory subunits of gglutamylcysteine synthetase were elevated approximately 2-fold. A 7fold increase in catalase expression in the resistant cell line indicated an adaptive response to oxidative stress. In MEF made from WT and GSTP1-1 KO mice, KO cells exhibited 2-fold resistance to TER286 compared to the WT [96.0 ±  20.0 mM versus 177.5 ± 42.0 mM]. These data support the rationale that tumors expressing high levels of GSTP1-1 will be more sensitive to cytotoxic effects of the drugs.

A photoactivatable glutathione-drug conjugate [³⁵S]-azidophenacyl-glutathione ([³⁵S]APA-SG) was synthesized and used to identify protein(s) involved in recognition and/or transport of glutathione conjugates of electrophilic drug species. An ~460 kDa protein was found to be highly labeled by [³⁵S]APA-SG in an adriamycin resistant HL60 (HL60/ADR) cell line and identified as the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by amino acid sequence analysis, Western blot analysis and by immunoprecipitation using specific antibodies. Binding specificity was confirmed by competition isotope dilution assays with purified proteins. A 15 to 20-fold increase in DNA-PKcs expression in HL60/ADR cell line was accompanied by an equivalent increase in [³⁵S]APA-SG binding. APA-SG, together with other glutathione conjugates and analogues inhibited the DNA-PK mediated phosphorylation of an in vitro peptide substrate in a concentration-dependent manner. Using different antibodies to immunoprecipitate the individual components of the DNA-PK complex (DNA-PKcs, Ku70 and Ku80), it was shown that APA-SG caused a destabilization of the trimeric holoenzyme complex by dissociating the catalytic subunit from the Ku heterodimer. These data suggest that the kinase-mediated signaling is inhibited when glutathione conjugates bind to DNA-PKcs, and may also indicate a possible strategy for design of novel DNA-PK inhibitors.

ATP binding cassette (ABC) transporters are a family of evolutionary conserved transmembrane proteins involved in the transport of a wide variety of substrates across membranes. We have recently identified and cloned a new human cDNA, ABC2, which maps to chromosome 9q34. The protein product has an apparent molecular weight of 230 kDa. ABC2 gene amplification has been linked to estramustine resistance in an ovarian carcinoma cell line; Western analysis confirmed increased ABC2 protein levels. Catalase levels were also increased in the estramustine-resistant cell line. Fractionation by differential centrifugation showed that ABC2 is enriched in the peroxisomal and lysosomal fractions. With triple immunofluorescence microscopy techniques, simultaneous localization of catalase and ABC2 was consistent with peroxisomal compartmentalization (Figure 1). Additionally, a fluorescently labeled dansylated estramustine appeared as a distinct vesicular staining pattern and also co-localized with the ABC2 and catalase immunofluorescence. These findings suggest that ABC2 is an integral peroxisomal membrane protein involved in the sequestration of estramustine and, by extrapolation, perhaps other estrogen based molecules.

dansylated estramustine anti-catalase antibody anti-ABC2 antibody

SKOV3                             SKOVEM15

FIGURE 1. Panels A, C, and E are wild type SKOV3 ovarian cancer cells; Panels B, D, and F are SKOVEM15 estramustine resistant cells. Panels A and B show the distribution of a fluorescently labeled dansylated estramustine (visualized at 360 nm.) Panels C and D are immunofluorescent images of anti-catalase (marker for peroxisomes) antibody with anti-sheep Cy2 secondary antibody. Panels E and F are immunofluorescent images of anti-ABC2 antibody and an anti-rabbit rhodamine secondary. The arrows represent areas of co-localization of ABC2, catalase and dansylated estramustine. There are more peroxisomes in the drug resistant cells. The scale bar is 10 mm.

PUBLICATIONS

ADLER, V, YIN, Z., FUCHS, S.Y., BENEZRA, M., ROSARIO, L., TEW, K.D., PINCUS, M.R., SARDANA, M., HENDERSON, C.J., WOLF, C.R., DAVIS, R., RONAI, Z. Regulation of JNK signaling by GSTp. EMBO J. 18:1321-1334, 1999.

GATE, L., SCHULTZ, M., WALSH, E., DHALLUIN, S., NGUYEN, BA, G., TAPIERO, H., TEW, K.D. Impact of dietary supplement of Crassotrea gigas extract (JCOE) on glutathione levels and glutathione S-transferase activity in rat tissues. In Vivo 12:299-304, 1998.

RUSCOE, J., O'BRIEN, M.L., FREER, S., SHEN, H., ROSARIO, L., VULEVIC, B., TEW, K.D. Co-ordinate expression of detoxification genes in drug resistance. Clin. Chem. Enzymol. Commun. (in press).

SANGRAJRANG, S., DENOULET, P., MILLOT, G., TATOUD, R., PODGORNIAK, M-P., TEW, K.D., CALVO, F., FELLOUS, A. Estramustine correlates with tau overexpression in human prostatic carcinoma cells. Int. J. Cancer 77:626-631, 1998.

SHEN, H., SCHULTZ, M.P., TEW, K.D. Glutathione conjugate interactions with DNA-dependent protein kinase. J. Pharmacol. Exp. Ther. (in press).

TAPIERO, H., GATE, L., DHALLUIN, S., NGUYEN BA, G., SOUPRAMANIEN, V., KOUYATE, J., TEW K.D. The antioxidant effects of Crassostrea gigas extract (JCOE) in human volunteers. In Vivo 12:305-310, 1998.

TEW, K.D., SHEN, H., LAING, N.M. Pleiotropic adaptations in drug resistance and stress response. In Progress in Cancer Therapy, Volume II, edited by G. Hortobagyi, D. Khayat. Blackwell Science, Massachusetts (in press).

^a   C.J. Henderson: ICRF Molecular Pharmacology Unit, University of Dundee, UK

^b   C.R.Wolf: ICRF Molecular Pharmacology Unit University of Dundee, UK

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998