Changes in radiation sensitivity and steroid receptor content induced by hormonal agents and ionizing radiation in breast cancer cells in vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM(R)-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM(R)-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM(R)-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM(R)-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

Sensitization to doxorubicin resistance in breast cancer cell lines by tamoxifen and megestrol acetate

Acquired drug resistance is a major factor in the failure of doxorubicin-based chemotherapy in breast cancer. We determined the ability of megestrol acetate and/or tamoxifen to reverse doxorubicin drug resistance in a doxorubicin-resistant breast cancer line (the human MCF-7/ADR). The cytotoxicity of doxorubicin, megestrol acetate, and/or tamoxifen was determined in the sensitive and resistant cell lines utilizing the sulphorhodamine B assay. Tamoxifen alone produced an IC50 (concentration resulting in 50% inhibition of control growth) of 10.6 microM, whereas megestrol acetate alone resulted in an IC50 of 48.7 microM in the MCF-7/ADR cell line. The IC50 of doxorubicin in MCF-7/ADR was 1.9 microM. Neither megestrol acetate alone nor tamoxifen alone at 1 or 5 microM altered the IC50 of doxorubicin. However, the combination of tamoxifen (1 or 5 microM) and megestrol acetate (1 or 5 microM) synergistically sensitized MCF-7/ADR cells. Additionally, megestrol acetate and tamoxifen inhibited iodoarylazidoprazosin binding to P-glycoprotein, and, in their presence, there was an increased doxorubicin accumulation in the MCF-7/ADR cells. Furthermore, the combination of tamoxifen and megestrol acetate had much less effect on the cytotoxicity of doxorubicin in MCF-7 wild-type cells. Clinically achievable concentrations of tamoxifen and megestrol acetate can largely sensitize MCF-7/ADR to doxorubicin. The combination of these three drugs in a clinical trial may be informative.     

Personal experience in the diagnosis and treatment of hepatic echinococcosis

A series of newly developed paclitaxel analogues have been tested for their growth inhibitory activity on two human breast cancer cell lines, one of which expresses the MDR (multidrug resistance) phenotype. Paclitaxel (taxol) was used as a reference compound. Three new classes of taxanes were analyzed: the cephalomannine compounds, the pyrazoline derivatives and the seco-derivatives. Our results demonstrated an increased antiproliferative activity of pyrazoline derivatives on drug-resistant cancer cells with respect to paclitaxel. These compounds were able to block MDR-bearing MCF-7 ADRr cells in the G2/M phase of cell cycle and, consequently, induce programmed cell death. In keeping with the antiproliferative effects, cells treated with paclitaxel derivatives showed a more pronounced cell cycle arrest than the parent compound paclitaxel. Also, apoptotic cell death, calculated as a percent of DNA fragmentation, occurred to a greater extent in cells exposed to pyrazoline derivatives. The development of new paclitaxel analogues with greater antitumour activity on MDR-positive cells may be useful in selecting new taxanes effective on resistant tumors.     

Induction of human breast cancer cell apoptosis from G2/M preceded by stimulation into the cell cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, A(II)) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of A(II). MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 microM A(II) when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1-2 Mb fragments appeared after 6 hr, while 30-50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30-50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that A(II) blocks MDA-MB-231 cells in G2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. A(II) demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, "normal" human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that A(II) caused stimulation into S phase, and that G2/M was the phase of the cell cycle from which cells apoptosed. A(II) caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited A(II)-induced cell death, indicating that its toxicity requires de novo protein synthesis.     

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.     

In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGFbeta1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGFbeta1 neutralizing antibody confirms the correlation between induction of active TGFbeta1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGFbeta1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGFbeta1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Schedule-dependent interaction of doxorubicin, paclitaxel and gemcitabine in human breast cancer cell lines

We showed previously that a sequential treatment with doxorubicin (4 hr) followed by paclitaxel (24 hr) (Dox-->Pacl) induces a synergistic cytotoxic effect in the BRC-230 breast cancer cell line and in human primary breast cancer cultures. The validity of this experimental finding was confirmed in a clinical phase I/II study on advanced breast cancer patients. To improve the cytotoxic effect obtained by the Dox-->Pacl sequence, we analyzed the effect of adding gemcitabine (Gem) to the Dox-->Pacl sequence in a preclinical study. Our study was performed on BRC-230 and MCF-7 cell lines, and cytotoxic activity was evaluated by the sulforhodamine B assay and the type of drug interaction by Drewinko's test. When Gem (0.01 microg/ml for 24 hr) was given immediately or 24 hr after Dox-->Pacl, an antagonistic cytotoxic effect was observed. Conversely, a synergistic effect was found when Gem was given 48 hr after Dox-->Pacl. From results of flow cytometric analysis, the synergistic effect was attributed to cell cycle perturbation. Cells were arrested in G2-M (95% in treated vs. 21% in control samples) 24 hr after Dox-->Pacl treatment. The block progressively recovered thereafter, and after a further 24 hr, at the time of Gem treatment, the cells progressed into the G1-S phase boundary (the cell cycle phase susceptible to the cytocidal effect of the drug). Our findings suggest that the interactions of Dox, Pacl and Gem are highly schedule- and time-dependent and should be taken into consideration in the planning of clinical protocols.     

Effects of OK-432 (picibanil) on the estrogen receptors of MCF-7 cells and potentiation of antiproliferative effects of tamoxifen in combination with OK-432

OK-432 (picibanil), a streptococcal preparation, has a strong biological response modifier (BRM) function and is expected to produce clinical improvement and prolongation of survival in treated cancer patients in Japan. We were interested in whether OK-432 augments estrogen receptor (ER) levels in breast cancer. To investigate the effect of the BRMs on cellular growth and the characteristics of ER and progesterone receptors (PgR) in the human breast cancer cell line MCF-7, we used OK-432, Krestin (PSK), a protein-bound polysaccharide extracted from Coriolus versicolor, and lentinan, a fungal branched (1...3)-beta-D-glycan. OK432 and PSK dose dependently inhibited DNA synthesis of MCF-7 cells, and the 50% inhibitory concentrations of OK-432 and PSK were 1.2 KE (klinische Einheit, clinical unit)/ml and 200 micrograms/ml, respectively. Lentinan showed no direct anticancer effect in vitro. We found that OK-432 induced a 2-fold increase in ER levels in MCF-7 cells at 0.005 KE/ml, but not in PgR. Lentinan and low-dose PSK did not change ER or PgR levels, but high-dose PSK decreased ER and PgR. We also studied the combined effect of OK-432 and antiestrogens, tamoxifen (TAM) and DP-TAT-59. The combined treatment with OK-432 and TAM showed an additive inhibitory effect on MCF-7 cells. These results suggest that OK-432 may augment the therapeutic effect of TAM in breast cancer.     

Regulation of the function of P-glycoprotein by epidermal growth factor through phospholipase C

Many multidrug-resistant (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp). However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear. Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC. Treatment of the human MDR breast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20-50%. The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Treatment of MDR cells with EGF also had detectable effects on P-gp function. For example, following incubation of MCF-7/AdrR cells with ECF, we observed a consistent decrease in total vinblastine (VBL) accumulation. Kinetic analysis revealed this change to be due to an increase in membrane efflux. The latter was measured by the initial uptake velocity, which was inhibited by EGF. VBL uptake measured at 0-320 sec was inhibited by 20-40%, which was associated with a similar increase in VBL efflux. EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells. These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC.     

Dietary iron overload as a risk factor for hepatocellular carcinoma in Black Africans

We analyzed the effect of high temperature (a 1-h incubation at 43 degrees C) on the accumulation and cytotoxicity of vinblastine and docetaxel in two model cell lines, K562 and MESSA, and their multidrug resistance (MDR) counterparts, K562/R7 and MESSA/Dx5. High temperature increased the amount of intracellular vinblastine and docetaxel significantly in MESSA cell and, to a much lesser extent, in K562 cells. MDR-positive cells retained a profound drug accumulation defect at 43 degrees C. Hyperthermia enhanced the cytotoxic effect of vinblastine (but not docetaxel) in both K562 and MESSA cells, but not in the MDR-positive variants. PSC833, a potent modulator of P-glycoprotein, induced high levels of drug accumulation in the two MDR-positive cell lines at both 37 degrees C and at 43 degrees C. PSC833 also significantly reduced the resistance levels of the two MDR-positive lines at both 37 degrees C and at 43 degrees C. The effect of hyperthermia on drug accumulation thus seems to depend on the cell line, whereas the effect on cytotoxicity depends on the type of compound. The MDR phenotype remains a therapeutic obstacle at 43 degrees C but is accessible to modulation.     

In vitro effect of GF120918, a novel reversal agent of multidrug resistance, on acute leukemia and multiple myeloma cells

Resistance to chemotherapy in multiple myeloma (MM) and acute myeloid leukemia (AML) is frequently caused by multiple drug resistance (MDR), characterized by a decreased intracellular drug accumulation. MDR is associated with expression of P-glycoprotein (P-gp). GF120918, an acridine derivative, enhances doxorubicin cell kill in resistant cell lines. In this study, the effect of GF120918 on MDR cell lines and fresh human leukemia and myeloma cells was investigated. The reduced net intracellular rhodamine-123 (Rh-123) accumulation in the MDR cell lines RPMI 8226/Dox1, /Dox4, /Dox6 and /Dox40 as compared with wild-type 8226/S was reversed by GF120918 (0.5-1.0 microM), and complete inhibition of rhodamine efflux was achieved at 1-2 microM. This effect could be maintained in drug-free medium for at least 5 h. GF120918 reversal activity was significantly reduced with a maximum of 70% in cells incubated with up to 100% serum. GF120918 significantly augmented Rh-123 accumulation in vitro in CD34-positive acute leukemia (AML) blasts and CD38-positive myeloma (MM) plasma cells obtained from 11/27 de novo AML and 2/12 refractory MM patients. A significant correlation was observed between a high P-gp expression and GF120918 induced Rh-123 reversal (P=0.0001). Using a MRK16/IgG2a ratio > or = 1.1, samples could be identified with a high probability of GF120918 reversal of Rh-123 accumulation. In conclusion, GF120918 is a promising MDR reversal agent which is active at clinically achievable serum concentrations.     

The role of mechanical and neural restraints to joint range of motion during passive stretch

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor-independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF-7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme-specific membrane association of PKC-epsilon, which was time-dependent (as early as 5 min post-treatment) and dose-dependent (5.0-20 microM). Tamoxifen did not influence translocation of alpha, beta, gamma, delta or zeta PKC isoforms. Structure-activity relationship studies demonstrated chemical requirements for PKC-epsilon translocation, with tamoxifen, 3-OH-tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N-dimethyl-2-[(4-phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent-induced PKC-epsilon translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC-epsilon has been associated with cell differentiation and cellular growth-related processes, the antiproliferative influence of tamoxifen on MCF-7 cells may be related to the interaction with PKC-epsilon.     

Anti-proliferative effect of toremifene and tamoxifen on estrogen receptor-lacking anaplastic thyroid carcinoma cell lines

The antitumor effects of toremifene were studied in vitro using 10 anaplastic thyroid carcinoma cell lines and were compared with those of tamoxifen. The antitumor effect of toremifene was dose-dependent and the IC50 values for these cell lines ranged 20.1-58.5 microM. Although the cell lines expressed multidrug resistance gene mRNA, multidrug resistance-associated protein gene mRNA and insulin-like growth factor-1 (IGF-1) receptor mRNA to various degrees, nine of them lacked estrogen receptor expression as evaluated by immunocytochemistry. These data indicate that toremifene, as well as tamoxifen, is cytolytic at relatively high doses for multidrug-resistant anaplastic thyroid carcinoma cell lines.     

The estrogen receptor from a tamoxifen stimulated MCF-7 tumor variant contains a point mutation in the ligand binding domain

The nonsteroidal antiestrogen tamoxifen (TAM) is the most commonly used endocrine treatment for all stages of breast cancer in both pre- and postmenopausal women. However, the development of resistance to the drug is common, as most patients treated with TAM eventually experience a recurrence of tumor growth. One of the potential mechanisms of treatment failure is the acquisition by the tumor of the ability to respond to TAM as a stimulatory rather than inhibitory ligand. We (Gottardis and Jordan, Cancer Res 48:5183-5187, 1988; Wolf et al., J Natl Cancer Inst 85:806-812, 1993) and others (Osborne et al., Eur J Cancer Clin Oncol 23: 1189-1196, 1987; Osborne et al., J Natl Cancer Inst 83: 1477-1482, 1991) have extensively described the reproducible development of TAM stimulated growth in a laboratory model system using MCF-7 human breast cancer cells grown as solid tumors in athymic mice. In this paper we report on the isolation of an estrogen receptor (ER) from a TAM stimulated tumor (MCF-7/MT2) which contains a point mutation that causes a tyrosine for aspartate substitution at amino acid 351 in the ligand binding domain. The mutant appears to the major form of ER expressed by this tumor. We also report that only wild type ER was detected in three other TAM stimulated MCF-7 tumor variants, suggesting that multiple mechanisms are possible for the development of TAM stimulated growth. The implications of these findings are discussed.     

Sequence determination and molecular characterization of gigantin, a cytotoxic protein produced by the mould Aspergillus giganteus IFO 5818

To examine the ability of estrogens and anti-estrogens to induce cellular transformation and genetic effects, Syrian hamster embryo (SHE) cells were treated with estrogens, 17beta-estradiol (E2) or diethylstilbestrol (DES), or with anti-estrogens, tamoxifen (TAM), toremifene (TOR) or ICI 164,384. Treatment with each substance for 1-3 days suppressed cellular growth in a dose-dependent manner. Colony-forming efficiency (CFE) increased following treatment of cells with E2 or DES for 48 hr at 3 or 10 microM but decreased at 20 or 30 microM. In contrast, CFE was increased by treatment with TAM, TOR or ICI 164,348 over the concentration range examined (1-30 microM). Treatment with each chemical at 1-30 microM for 48 hr caused morphological transformation of SHE cells in a dose-related fashion. The highest frequency was exhibited in SHE cells treated with DES at 20 microM and was 2 times higher than that induced by treatment with benzo[alpha]pyrene (B[alpha]P) at 4 microM. Transformation frequencies induced by other substances (E2, TAM, TOR and ICI 164,348) did not exceed that induced by the B[alpha]P treatment. TOR showed a higher transforming ability over all concentrations examined when compared to the other anti-estrogens (TAM and ICI 164,348). No significant increases in the frequencies of chromosomal aberrations were observed in SHE cells that were treated with any of the chemicals. However, treatment of SHE cells with each chemical induced a dose-dependent increase of aneuploid cells in the near diploid range. Our results indicate that the ability of the estrogens and anti-estrogens to induce numerical chromosomal abnormality may be involved in their cell transformation activity and potential carcinogenicity.     

Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel

The epothilones are naturally occurring, cytotoxic macrolides that function through a paclitaxel (Taxol)-like mechanism. Although structurally dissimilar, both classes of molecules lead to the arrest of cell division and eventual cell death by stabilizing cellular microtubule assemblies. The epothilones differ in their ability to retain activity against multidrug-resistant (MDR) cell lines and tumors where paclitaxel fails. In the current account, we focus on the relationship between epothilone and paclitaxel in the context of tumors with multiple drug resistance. The epothilone analogue Z-12,13-desoxyepothilone B (dEpoB) is >35,000-fold more potent than paclitaxel in inhibiting cell growth in the MDR DC-3F/ADX cell line. Various formulations, routes, and schedules of i.v. administration of dEpoB have been tested in nude mice. Slow infusion with a Cremophor-ethanol vehicle proved to be the most beneficial in increasing efficacy and decreasing toxicity. Although dEpoB performed similarly to paclitaxel in sensitive tumors xenografts (MX-1 human mammary and HT-29 colon tumor), its effects were clearly superior against MDR tumors. When dEpoB was administered to nude mice bearing our MDR human lymphoblastic T cell leukemia (CCRF-CEM/paclitaxel), dEpoB demonstrated a full curative effect. For human mammary adenocarcinoma MCF-7/Adr cells refractory to paclitaxel, dEpoB reduced the established tumors, markedly suppressed tumor growth, and surpassed other commonly used chemotherapy drugs such as adriamycin, vinblastine, and etoposide in beneficial effects.     

Retroviral transfer of deoxycytidine kinase into tumor cell lines enhances nucleoside toxicity

Deoxycytidine kinase (dCK) phosphorylates a number of nucleoside analogues that are useful in the treatment of various malignancies. Although the level of dCK activity in malignant cells is thought to correlate with chemotherapeutic response, no direct data are available to support this assumption. We have tested this hypothesis by infecting three tumor cell lines, MCF-7, HT-29, and H1437, with the retroviral vector LNPO containing either dCK or LacZ cDNA and measuring the corresponding sensitivity to nucleoside analogues. DCK activity was increased by 1.7-, 2.3-, and 16-fold in MCF-7, HT-29, and H1437 cells, respectively. Northern and Western blots demonstrated a similar increase in mRNA and protein levels. As a result of dCK expression, MCF-7 cells demonstrated a 2.5-fold increase in drug sensitivity to 1-beta-D-arabinofuranosylcytosine (AraC) and 2-chloro-2'-deoxyadenosine (CdA). HT-29 cells had a 7-fold increase in sensitivity to AraC, CdA, and 2-fluoro-9-beta-D-arabinofuranosyladenine, whereas H1437 cells demonstrated a 20- to 106-fold increase. For all three drugs, there was a linear relationship between dCK activity in clonally derived cell lines and IC50s. These data demonstrate a direct effect of dCK activity on drug sensitivity in cell lines. Because many tumors have relatively low levels of dCK, it is possible that dCK gene transfer will be a useful adjunct to the treatment of these malignancies.