Comparison of the properties of human breast cancer cells: MCF-7 and MCF-7 cells selected for resistance to etoposide.
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Antineoplastic Agents↗
Biomedical subjects
J G Cory
Publications and source records attributed to J G Cory.
Phenotypic changes in mouse leukemia L1210 cells with alterations in the effector-binding subunit of ribonucleotide reductase.
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Animals↗
Altered sensitivity of deoxyadenosine-resistant mouse leukemia L1210 cells to apoptosis induced by 7-hydroxystaurosporine.
The deoxyadenosine-resistant mouse leukemia L1210 cell line (Y8) has previously been shown to be more sensitive to apoptosis induced by DNA damaging agents and by protein synthesis inhibitors than the parental wild-type L1210 (WT) cells. These responses occur independently of p53 as both cell lines lack wild-type p53 function. Recent evidence suggests that a serine/threonine kinase is involved in the divergent cellular responses of the WT and Y8 cells. In the present study, the effects of 7-hydroxystaurosporine (UCN-01), a relatively specific serine/threonine kinase inhibitor, were examined in the WT and Y8 cells. Both cell lines were equally sensitive to the growth inhibitory effects of UCN-01. However, the Y8 cells accumulated in G0/G1 and became apoptotic. Apoptosis induced by UCN-01 in the Y8 cells was mediated by a caspase-3-like activity which could be partially blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. UCN-01 did not alter the phosphorylation status of cdc2 nor cyclin B1 and cdc2 protein levels in either cell line.
Alkaloids↗
Lack of competition of substrates for P-glycoprotein in MCF-7 breast cancer cells overexpressing MDR1.
The MCF-7/Adr cells overexpress MDR-1 which contributes to the drug-resistant phenotype. Our studies show: 1. The retention of daunomycin in the MCF-7/Adr cells relates to a temperature-dependent and energy-dependent process. 2. The MCF-7/Adr cells retain less rhodamine-123 than the parental MCF-7 cells. 3. The MCF-7/Adr cells retain less daunomycin than the parental MCF-7 cells as measured by mean fluorescence or radioactive daunomycin. 4. Cyclosporin A and verapamil effectively block the effluxes of rhodamine-123 and daunomycin from the MCF-7/Adr cells. 5. On short-term incubation, 2-deoxyglucose lowers the NTP levels to a greater extent than sodium azide, showing the importance of glycolysis in the MCF-7 cell lines. 6. Although the MCF-7/Adr cells show cross-resistance to VP-16, actinomycin D and vinblastine, these drugs do not compete with daunomycin for the efflux pump. 7. These data suggest that either there must be multiple MDR-1 pumps that differ in substrate specificity or that there are distinct substrate sites on MDR-1.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP): an inhibitor of ribonucleotide reductase with antineoplastic activity.
The enzyme RR catalyzes the conversion of ribonucleoside diphosphates to their deoxyribonucleotide counterparts. RR is critical for the generation of the cytosine, adenine, and guanine deoxyribonucleotide 5'-triphosphate building blocks of DNA, which are present in cells as exceedingly small intracellular pools. Therefore, interference with the function of RR might well result in an agent with significant antineoplastic activity, particularly against rapidly proliferating tumor cells. HUr is the only inhibitor of RR in clinical usage; this agent, however, is a relatively poor inhibitor of the enzyme and has a short serum half-life. Consequently, HUr is a relatively weak anticancer agent. In an effort to develop a more potent inhibitor of RR with utility as an anticancer agent, we have synthesized 3-AP and demonstrated (a) potent inhibition of L1210 leukemia cells in vitro, (b) curative capacity for mice bearing the L1210 leukemia, (c) marked inhibition of RR, and (d) sensitivity of HUr-resistant cells to 3-AP. These findings collectively demonstrate the clinical potential of 3-AP as an antineoplastic agent.
Animals↗
Effect of doxorubicin on wild-type and deoxyadenosine-resistant mouse leukemia L1210 cells.
Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of the mRNA and protein for p53. However, the p53 expressed by the wild-type cells is a mutant form of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT L1210 cells does not maintain constitutive levels of p53 mRNA or protein. Upon DNA damage, induced by doxorubicin, neither the WT nor the Y8 cells block in G0/G1; the cells block in G2/M. However, treatment of the Y8 cells with doxorubicin results in a much greater fraction of cells becoming apoptotic compared to the WT cells. Doxorubicin treatment resulted in the induction of p53 mRNA in the WT cells, but not the Y8 cells. WAF1, c-myc and Bax mRNAs were also induced by doxorubicin in the WT cells but not in the Y8 cells. The constitutive levels of WAF1 and Gadd45, unexpectedly seen in the p53-deficient Y8 cells, decreased following doxorubicin treatment. The comparison of the effects of DNA damage, as measured by mRNA levels, induced by X-irradiation or doxorubicin were found to vary between the WT and Y8 cells and for the particular mRNA studied. The effect of doxorubicin or X-irradiation on the cell cycle could be overridden in the WT cells by caffeine. Comparisons of DNA damage induced by doxorubicin or X-irradiation show that although the Y8 cells are more sensitive to these damaging agents than the WT cells, the effects on gene expressions are not identical.
Animals↗
Mouse leukemia L1210 cells selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone show altered response to DNA damaging agents.
An L1210 cell line (MQ-580) selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ), had been previously shown to have altered properties related to the non-heme iron subunit of ribonucleotide reductase (RR). In addition, the MQ-580 cells had other metabolic alterations that were consistent with multidrug resistance. Neither the wild-type (WT) nor the MQ-580 cells showed a G0/G1 block in response to X-irradiation. Since the MQ-580 cells also showed resistance to adriamycin (ADR), the WT cells were more sensitive to the effects of ADR than the MQ-580 cells. However, when the MQ-580 cells were treated with the combination of ADR plus verapamil, the MQ-580 cells showed cellular responses that included cell cycle block in G2/M and increased apoptosis. The WT cells, in response to the combination of ADR plus verapamil, blocked in the S-phase of the cell cycle with an increased necrotic cell population in comparison to treatment with ADR alone. While MAIQ could be shown to cause an apoptotic response in the WT and MQ-580 cells, the concentrations of MAIQ required to induce apoptosis were 20- to 40-times the IC50 value. However, even though the MQ-580 cells were 8-fold more resistant to MAIQ than the WT cells, the MQ-580 cells were more sensitive to MAIQ-induced apoptosis. These data show that alterations at the RR site lead to phenotypic expressions not overtly related to RR. However, because of the critical role that RR plays in cell division and DNA repair, the changes observed with respect to cell cycle and apoptosis, may in fact, be a direct consequence of the alteration at RR.
Animals↗
Cellular responses in mouse leukemia L1210 cells made resistant to deoxyadenosine.
Recent studies have implicated nucleotides in diverse and unexpected functions related to p53 levels, p53-dependent G0/G1 cell cycle arrest, and the role of dATP in the activation of the caspase-induced apoptosis. Using deoxyadenosine-resistant L1210 cells (ED2 and Y8) that had ribonucleotide reductase that was not sensitive to inhibition by dATP and also exhibited other metabolic alterations, the properties of these cells with respect to the role(s) of nucleotides in these functions were explored. In the ED2 and Y8 cells that did not express p53 protein, the pools of UTP, CTP, ATP, and GTP were markedly decreased. The decreased cellular levels of UTP and CTP did not result in these cells being more sensitive to either PALA or acivicin. The ED2 and Y8 cells did not block in G0/G1 in response to PALA treatment even though the basal cellular concentrations of UTP and CTP were reduced 50 to 80%. While it has been shown that dATP in combination with cytochrome c is involved in the apoptotic pathway, the concentration of exogenous deoxyadenosine required to induce apoptosis in the parental L1210 cells was far in excess of the concentration required to inhibit cell growth. Deoxyadenosine did not cause an increase in apoptosis in the deoxyadenosine-resistant Y8 cells. These data suggest that the new roles ascribed to nucleotides may be specific for the particular cell type under very specific conditions.
Animals↗
Multifactorial mechanisms of drug resistance in tumor cell populations selected for resistance to specific chemotherapeutic agents.
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Animals↗
Effect of ionizing radiation on wild-type and mutant mouse leukemia L1210 cells.
Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of p53 mRNA and protein. However, the p53 expressed by the wild-type L1210 cells was found to be a mutant form of p53 (relative to normal mouse fibroblast p53 sequence) having a point mutation in the DNA binding domain of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT cells had previously been shown to lack the expression of p53 but to respond to cycloheximide (CHX) treatment by superinduction of p53 mRNA. The mRNA for p53 induced by CHX had the same sequence as the p53 from normal mouse fibroblasts. Although the Y8 cells had no constitutive levels of p53 mRNA or protein, the Y8 cells expressed constitutive levels of WAF1 mRNA and protein. Gadd45 mRNA was also present in the Y8 cells. Subjecting the WT or Y8 cells to ionizing radiation did not result in a G0/G1 cell cycle block; the cells blocked in G2/M. The Y8 cells were much more sensitive to the irradiation treatment than the WT cells, resulting in marked increases in apoptosis in the Y8 cells. Although radiation treatment induced p53 mRNA, but no p53 protein, in the Y8 cells, WAF1 mRNA was induced in the Y8 cells. These data indicate that there are p53-independent pathway(s) that may still involve WAF1 and Gadd45 with respect to cell cycle control and apoptosis.
Animals↗
Overexpression of the multidrug resistance genes mdr1, mdr3, and mrp in L1210 leukemia cells resistant to inhibitors of ribonucleotide reductase.
L1210 MQ-580 is a murine leukemia cell line resistant to the cytotoxic activity of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone class of inhibitors of ribonucleotide reductase. The line is cross-resistant to etoposide, daunomycin, and vinblastine. L1210 MQ-580 cells expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a relatively newly developed inhibitor of ribonucleotide reductase. The accumulation of [14C]3-AP by L1210 MQ-580 cells was 5- to 6-fold less than by parental L1210 cells. An increased rate of efflux of 3-AP was responsible for the lower steady-state concentration of 3-AP in resistant cells. In reverse transcription-polymerase chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 gene, compared with parental L1210 cells. Measurement of the steady-state concentration of doxorubicin, a potential substrate for both the mdr and mrp gene products, demonstrated that L1210 MQ-580 cells accumulated 4-fold less anthracycline than parental cells. These findings indicate that drug efflux is a major determinant of the pattern of cross-resistance of L1210 MQ-580 cells. To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effects of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively. The transfectants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less [14C]3-AP than their parental mock-transfected counterparts. Moreover, the cytotoxic activity of 3-AP was significantly greater in two double mrp gene knockout cell lines than in parental W 9.5 embryonic stem cells. Thus, the results suggest that 3-AP is a substrate for both the P-glycoprotein and MRP and that baseline MRP expression has the capacity to exert a protective role against the toxicity of this agent.
ATP Binding Cassette Transporter, Subfamily B↗
Alterations in the properties of mouse leukemia L1210 cell lines selected by different methods for resistance to deoxyguanosine.
Mouse leukemia L1210 cells were generated for resistance to deoxyguanosine by two different methods. In one case the L1210 cells were subjected to gradual increases in deoxyguanosine (dGuo-R); in the second approach, the cells were subjected to deoxyguanosine at a concentration ten times the IC50 value and plated out on soft agar (D-92). The dGuo-R and D-92 cell lines had different phenotypic expressions. The dGuo-R cells showed a higher degree of resistance to dGuo than the D-92 cells. The levels of resistance to other cytotoxic drugs such as araC or 2-chloro-2'-deoxyadenosine did not necessarily correlate with the degree of resistance to dGuo. Deoxycytidine kinase activity was decreased in both of the cell lines, although there was a larger decrease in the dGuo-R cell line. The levels of kinase activities toward the other substrates were not all coordinately decreased in these cell lines. The degree of resistance of these cell lines to dGuo cannot be ascribed solely to an alteration at the deoxycytidine kinase site.
Animals↗
Cellular delivery of nucleoside diphosphates: a prodrug approach.
PURPOSE: This study is concerned with cellular delivery/generation of 2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CDP), potent inhibitors of ribonucleotide reductase. It characterizes the phosphorylation steps involved in the conversion of 2'-azido-2'-deoxyuridine (N3Urd) and 2'-azido-2'-deoxycytidine (N3Cyd) to the corresponding diphosphates and explores a prodrug approach in cellular delivery of the inhibitor which circumvents the requirement of deoxynucleoside kinases. METHODS: Cell growth of CHO and 3T6 cells of known deoxycytidine kinase level was determined in the presence of N3Urd and N3Cyd. Activity of ribonucleotide reductase was determined in the presence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monophosphate of N3Urd was prepared and its biological activity was evaluated with CHO cells as well as with cells transfected with deoxycytidine kinase. RESULTS: N3Urd failed to inhibit the growth of both cell lines, while N3Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kinase levels found in the cells. Importance of the kinase was further established with the finding that the nucleoside analogs were inactive as reductase inhibitors in a permeabilized cell assay system while their mono- and di-phosphates were equally active. The prodrug was active in cell growth inhibition regardless of the deoxycytidine kinase level. CONCLUSIONS: The azidonucleosides become potent inhibitors of the reductase by two sequential phosphorylation steps. The present study indicates that the first step to monophosphate is rate-limiting, justifying a prodrug approach with the monophosphate.
Animals↗
Synthesis and biological activity of 3- and 5-amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone.
A series of 3- and 5-alkylamino derivatives, as well as other structurally modified analogues of pyridine-2-carboxaldehyde thiosemicarbazone, have been synthesized and evaluated as inhibitors of CDP reductase activity and for their cytotoxicity in vitro and antineoplastic activity in vivo against the L1210 leukemia. Alkylation of 3- and 5-amino-2-(1,3-dioxolan-2-yl)pyridines (1, 2) resulted in corresponding 3-methylamino, 5-methylamino, 3-allylamino, 5-ethylamino, 5-allylamino, 5-propylamino, and 5-butylamino derivatives (5, 6, and 11-15), which were then condensed with thiosemicarbazide to yield the respective thiosemicarbazones (7, 8, and 16-20). Oxidation of 3,5-dinitro-2-methylpyridine (21) with selenium dioxide, followed by treatment with ethylene glycol and p-toluenesulfonic acid, produced the cyclic ethylene acetal, 23. Oxidation of 2-(1,3-dioxolan-2-yl)-4-methyl-5-nitropyridine (26) with selenium dioxide, followed by sequential treatment with sodium borohydride, methanesulfonyl chloride, and morpholine afforded the morpholinomethyl derivative 30. Catalytic hydrogenation of 23 and 30 with Pd/C yielded the corresponding amino derivatives 24 and 31. Catalytic hydrogenation of 5-cyano-2-methylpyridine (33) with Raney nickel, followed by treatment with acetic anhydride, gave the amide derivative 35. N-Oxidation of 35, followed by rearrangement with acetic anhydride, produced the acetate derivative, 5-[(acetylamino)methyl]-2-(acetoxymethyl)pyridine (37). Repetition of the N-oxidation and rearrangement procedures with compound 37 yielded the diacetate derivative 39. Condensation of compounds 24, 31, and 39 with thiosemicarbazide afforded the respective 3,5-diaminopyridine-, 4-(4-morpholinylmethyl)-5-aminopyridine-, and 5-(aminomethyl)pyridine-2-carboxaldehyde thiosemicarbazones (25, 32, and 40). The most biologically active compounds synthesized were the 5-(methylamino)-, 5-(ethylamino)-, and 5-(allylamino)pyridine-2-carboxaldehyde thiosemicarbazones (8, 17, and 18), which were potent inhibitors of ribonucleotide reductase activity with corresponding IC50 values of 1.3, 1.0, and 1.4 microM and which produced significant prolongation of the survival time of L1210 leukemia-bearing mice, with corresponding optimum % T/C values of 223, 204, and 215 being obtained when administered twice daily for six consecutive days at dosages of 60, 80, and 80 mg/kg, respectively.
Animals↗
Differences in the properties of mammalian ribonucleotide reductase toward its substrates.
These studies, using three different reagents, show that the substrate properties of ribonucleotide reductase are specific but can be variable depending upon the nature of the interaction of the reagent with the holoenzyme or the individual subunit. Etheno-CDP, which acts as a competitive inhibitor with respect to CDP, interacts with the active site of the holoenzyme. This interaction was the result of rather tight structural requirements as epsilon-ADP did not result in a similar level of inhibition of either CDP or ADP reductase activities. The YL 1/2 antibody which binds very tightly to the NHI subunit has a much greater effect on CDP reductase activity than ADP reductase activity. The nonapeptide that corresponds to the C-terminus amino acid sequence of the NHI subunit and which binds to the EB subunit and aborts the formation of the NHI-EB active complex has a greater effect on ADP reductase activity than on CDP reductase activity. The use of reagents such as these can be helpful in dissecting the subtle but important differences in the substrate properties of mammalian ribonucleotide reductase.
Adenylyl Imidodiphosphate↗
Characterization of mouse leukemia L1210 cells resistant to the ribonucleotide reductase inhibitor 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone.
Mouse leukemia L1210 cells were generated for resistance to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ), a potent inhibitor of ribonucleotide reductase that is directed in the nonheme iron subunit (NHI) of the enzyme. The resistant cells, MQ-580, showed an 8-fold increase in IC50 toward MAIQ, a 4-fold increase in IC50 toward hydroxyurea, and also showed resistance to other ribonucleotide reductase inhibitors. In addition, the MQ-580 cell line was resistant to nonribonucleotide reductase inhibitors such as etoposide, daunomycin and vinblastine, but not to cisplatin. The mRNA for the NHI subunit was increased 7-fold in the MQ-580 cells with essentially no change in the mRNA level for the effector-binding subunit. The ribonucleotide reductase activity in the cell-free extracts prepared from the MQ-580 cells was only slightly elevated (30%). However, passage of the cell-free extract from the MQ-580 cells over Sephadex G-25 resulted in a 4.8-fold increase in specific activity over that of the wild-type cells. While the reductase activity in the cell-free extract from the MQ-580 cells did not show altered sensitivity to MAIQ, the reductase activity in the cell-free extract from the MQ-580 cells was much more sensitive to the effects of the iron-chelating agents Desferal and EDTA. The cell pellets from the MQ-580 cells were much darker in color than the pellets from the wild-type cells or hydroxyurea-resistant cells. The supernatant fraction from the MQ-580 cells after-SDS-PAGE showed the appearance of a strong Coomassie blue-staining band at 50 kDA that was not apparent in either the wild-type or hydroxyurea-resistant cells. This new resistant cell line offers an opportunity to explore differences in resistance mechanisms of drugs (e.g. MAIQ and hydroxyurea) that are directed at the same subunit of ribonucleotide reductase.
Animals↗
Structure-function relationships for a new series of pyridine-2-carboxaldehyde thiosemicarbazones on ribonucleotide reductase activity and tumor cell growth in culture and in vivo.
The synthesis of a new series of pyridine-2-carboxaldehyde thiosemicarbazones (HCTs) that have amino groups in the 3- and 5-positions has allowed the comparison of the structure/function relationships with regard to inhibition of ribonucleotide reductase activity, L1210 cell growth in culture and L1210 leukemia in vivo. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazones are more active than the corresponding 3-hydroxy-derivatives. The 3-amino-2-pyridine carboxaldehyde thiosemicarbazones were also more active then the 5-amino-2-carboxaldehyde thiosemicarbazones in inhibiting ribonucleotide reductase activity and L1210 cell growth in culture and in vivo. N-Acetylation of the 3-amino derivative resulted in a compound that was much less active both in vitro and in vivo; N-acetylation of the 5-amino derivative did not alter the in vitro inhibitory properties, but did eliminate the antitumor properties in vivo. When the most active HCTs were studied in more detail, it was found that the incorporation of [3H]thymidine into DNA was inhibited completely without the inhibition of [3H]uridine incorporation into RNA. Further, the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA was inhibited by the HCTs without an effect on the incorporation of cytidine into RNA. These data support the conclusion that ribonucleotide reductase is the major site of action of these HCTs. The 3-aminopyridine-2-carboxaldehyde thiosemicarbazones emerge as strong candidates for development for clinical trials in cancer patients.
Acetylation↗
Inhibitors of ribonucleotide reductase. Comparative effects of amino- and hydroxy-substituted pyridine-2-carboxaldehyde thiosemicarbazones.
A new series of alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazones (HCTs) was studied for their effects on L1210 cell growth in culture, cell cycle transit, nucleic acid biosynthesis and ribonucleotide reductase activity. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) were the most active compounds tested with respect to inhibition of cell growth and ribonucleotide reductase activity. 5-Aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AP) and 4-methyl-5-aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AMP) were slightly less active. 3-AP, 3-AMP, 5-AP and 5-AMP inhibited the incorporation of [3H]thymidine into DNA without affecting the rate of incorporation of [3H]uridine into RNA. The uptake and incorporation of [14C]cytidine into cellular ribonucleotides and RNA, respectively, were not decreased by 3-AP or 3-AMP; however, the incorporation of cytidine into DNA via ribonucleotide reductase was inhibited markedly. Thus, a pronounced decrease in the formation of [14C]deoxyribonucleotides from radioactive cytidine occurred in the acid-soluble fraction of 3-AP- and 3-AMP-treated L1210 cells. Consistent with an inhibition of DNA replication that occurred at relatively low concentrations of 3-AP and 3-AMP, cells gradually accumulated in the S-phase of the cell cycle; at higher concentrations of 3-AP and 3-AMP, a more rapid accumulation of cells in the G0/G1 phase of the cell cycle occurred, with the loss of the S-phase population, implying that a second less sensitive metabolic lesion was created by the HCTs. N-Acetylation of 3-AMP resulted in a compound that was 10-fold less active as an inhibitor of ribonucleotide reductase activity and 8-fold less active as an inhibitor of L1210 cell growth. N-Acetylation of either 5-AP or 5-AMP did not alter the inhibitory properties of these compounds. The results obtained provide an experimental rationale for the further development of the HCTs, particularly 3-AP and 3-AMP, as potential drugs for clinical use in the treatment of cancer.
Animals↗