Increased sensitivity to sodium salicylate-induced apoptosis in drug-resistant leukemia L1210 cells.

Mouse leukemia L1210 cells selected for resistance to deoxyadenosine contain ribonucleotide reductase that is not feedback inhibited by dATP. These deoxyadenosine-resistant cells (Y8) also do not express p53 protein but do have WAF1 and Gadd45 mRNA and protein. The Y8 cells show increased sensitivity to DNA damaging agents and kinase inhibitors. In these studies we show that in the presence of sodium salicylate (NaSal), the parental wild-type (WT) cells block in G2/M phase of the cell cycle while the Y8 cells show a marked increased in the G0/G1 population of cells. The Y8 cells are more sensitive to apoptosis induced by NaSal than the WT cells. NaSal treatment causes the induction of caspase-3-like activity in Y8 cells but no induction of caspase-3 activity in the WT cells. The caspase inhibitor, Ac-DEVD-CHO, decreased the percentage of Y8 cells in the early apoptotic fraction, but this decrease was reflected by an increase in the percent of cells in the late apoptotic/necrotic fraction. SB20358, a p38-MAP kinase inhibitor did not protect the Y8 cells from NaSal-induced apoptosis indicating that the p38-MAP kinase pathway was not involved in the NaSal-induced apoptotic pathway in the p53-independent Y8 cells.

Augmentation of apoptosis responses in p53-deficient L1210 cells by compounds directed at blocking NFkappaB activation.

A mouse leukemia L1210 cell line (Y8) selected for resistance to deoxyadenosine was found to be deficient in the expression of p53 mRNA and protein while maintaining the expression of WAF1/p21 mRNA and protein even under basal conditions. The Y8 cells were shown to be more sensitive to apoptosis induced by a variety of agents when compared to the parental wild-type (WT) L1210 cells. Roscovitine, an inhibitor of cdk 2 and cdk5, was one of the agents that caused increased apoptosis in the Y8 cells through a pathway that ultimately involved the activation of caspase-3 activity. In these studies, the effects of leflunomide and parthenolide (drugs reported to alter the activation of NFkappaB in a variety of cell types) were studied for their cell cycle and apoptotic effects in WT and Y8 cells as single agents and in combination with roscovitine. Leflunomide at IC50 concentrations had little effect on the cell cycle distribution of either the WT or Y8 cells while at higher concentrations caused a G0/G1 block in Y8 cells. Parthenolide, at IC50 concentrations, caused a G0/G1 cell cycle block in the WT and Y8 cells but at higher concentrations caused a G2/M block in the Y8 cells. The combinations of leflunomide and roscovitine or parthenolide and roscovitine did not alter, in a significant way the cell cycle distribution of the Y8 cells. However, in the presence of the combinations of leflunomide and roscovitine or parthenolide and roscovitine there were large increases in the fraction of Y8 cells undergoing early apoptosis without a corresponding increase in the necrotic fraction of cells. These data show that combinations of agents directed at different pathways or different steps of pathways involved in apoptosis can cause the cells to reach an apoptotic threshold that results in synergistic apoptosis.

Enhanced roscovitine-induced apoptosis is mediated by a caspase-3-like activity in deoxyadenosine-resistant mouse leukemia L1210 cells.

The deoxyadenosine-resistant mouse leukemia L1210 cell line (Y8) has previously been shown to have phenotypic differences that appear to be unrelated to the altered properties observed at the level of ribonucleotide reductase (RR). One of these changes is that the Y8 cells do not express p53. In response to DNA damaging agents, x-irradiation and doxorubicin, both the parental wild-type L1210 (WT) and Y8 cells undergo G2/M arrest, which is consistent with cells lacking wild-type p53 function. However, Y8 cells are much more sensitive to apoptosis induced by these agents than WT cells. Previous studies have also shown that expression of certain genes involved in cell cycle regulation is different between WT and Y8 cells. Recent evidence suggests that a serine/threonine kinase is involved in the divergent cellular responses of these cells. In the present study, the effects of roscovitine, a cyclin-dependent kinase inhibitor, were examined on the WT and Y8 cells. The WT cells blocked in G2/M, whereas Y8 cells became apoptotic. Apoptosis induced by roscovitine in the Y8 cells was mediated by a caspase-3-like activity. NF kappa B was activated to a much greater extent by roscovitine in the WT cells than in Y8 cells. The data also indicate that cyclin B1/cdc2 plays a role in the divergent p53-independent G2/M block and apoptotic responses of the WT and Y8 cells, respectively. Several key factors such as cathepsin B, caspase-1, release of cytochrome c into the cytosol, TNF-alpha signaling, FasL/Fas signaling, c-myc overexpression, and E2F-1 overexpression and induction were shown not to be involved in the apoptotic pathway(s) in the Y8 cells.

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.

Apoptosis induced by inhibitors of nucleotide synthesis in deoxyadenosine-resistant leukemia L1210 cells that lack p53 expression.

An L1210 cell line (Y8) selected for resistance to deoxyadenosine contains ribonucleotide reductase that is not subject to inhibition by dATP. In addition, the Y8 cells have other phenotypic expressions that include increased sensitivity to apoptosis induced by various agents such as radiation, doxorubicin, anisomycin and roscovitine. The Y8 cells were found to be more sensitive to apoptosis induced by methotrexate (MTX), tiazofurin (TZ), deoxyguanosine (dGuo) and N-(phosphonoacetyl)-L-aspartate (PALA). Deoxyguanosine, at concentrations that did not cause apoptosis in the Y8 cells, prevented the apoptotic response of the Y8 cells to MTX and TZ. Deoxycytidine had no effect. Since caspase-3 activation is involved in apoptotic pathways, the effects of the caspase-3 inhibitor, Ac-DEVD-CHO, were studied on the dGuo-, MTX- or TZ-induced apoptosis in the Y8 cells. Ac-DEVD-CHO caused a marked decrease in the fraction of cells in the early phase of apoptosis. However, there was a corresponding increase in the fraction of cells in the late apoptotic/necrotic stages of cell death. This is in marked contrast to the dGuo-induced decrease in apoptosis seen in the MTX- and TZ-treated Y8 cells in which there were no increases in the late apoptotic/necrotic fraction of cells. These data show that alterations of nucleotide pools in the Y8 cells cause marked increases in the apoptotic response which may indicate that the Y8 cells are much more susceptible to the effects of misincorporation of nucleotides into DNA than are the parental WT L1210 cells.

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.

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.

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.

Altered sensitivity of deoxyadenosine-resistant mouse leukemia L1210 cells to various kinase inhibitors.

The deoxyadenosine-resistant mouse leukemia L1210 cell line (Y8) has previously been shown to have phenotypic differences that appear unrelated to the altered properties observed at the level of ribonucleotide reductase (RR). In response to various stress factors, the parental wild-type (WT) L1210 cell line undergoes cell cycle arrest; Y8 cells become apoptotic. These responses are p53-independent. Cell cycle regulation also appears different between the two cell lines, suggesting that Y8 cells are more apoptotic because of alterations in their cell cycle compared to WT cells. In order to study the relationships between cell cycle regulation and apoptosis, the effects of 2-aminopurine (2-AP), wortmannin, and PD98059, were studied on WT and Y8 cells. 2-AP induced G2/M block in both WT and Y8 cells with differences in G0/G1 and S phase contents between the two cell lines. Wortmannin induced G0/G1 block in Y8 cells, while exhibiting no effect on WT cells. PD98059 had no effect on the cell cycle of either WT or Y8 cells. In response to each inhibitor, Y8 cells underwent apoptosis to a much greater extent than the parental WT cell line. These data suggest that the specific pathways that converge on the cell cycle are altered and may be involved in the differences between a tumor cell to block in cell cycle or to undergo apoptosis.

p53-independent anisomycin induced G1 arrest and apoptosis in L1210 cell lines.

An L1210 cell line selected for resistance to deoxyadenosine (Y8) has been shown to have barely detectable levels of p53 mRNA and no measurable p53 protein in comparison to the parental mouse wild-type (WT) L1210 cells. We now show that the protein synthesis inhibitors, anisomycin and cycloheximide, arrest WT cells in the G1 phase of the cell cycle and induce apoptosis in Y8 cells via a p53-independent mechanism. There was a decrease in Rb phosphorylation but without the induction of WAF1 protein. Anisomycin treatment activated NF kappa B in the WT cells as early as 0.5 hr after treatment but did not activate NF kappa B in the Y8 cells. Cycloheximide was neither as potent as anisomycin in arresting WT cells at G1 and inducing apoptosis in Y8 cells, nor as potent in decreasing Rb phosphorylation. The finding that caffeine could override the G1 arrest induced by anisomycin and cycloheximide in the WT cells further supports the idea that the effects of anisomycin or cycloheximide on the cell cycle are at the level of cell cycle regulation, and are likely not mediated exclusively through the inhibition of protein synthesis.

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.

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.

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.

Altered efflux properties of mouse leukemia L1210 cells resistant to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone.

A mouse leukemia L1210 cell line, denoted MQ-580, that was selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ), in addition to having altered properties at the ribonucleotide reductase site had other alterations that contributed to its resistant phenotype; these included the expression of p-glycoprotein and the multi-drug resistance associated protein (MRP). The efflux of rhodamine 123 (Rh-123) or daunomycin (Dau) was greatly increased in MQ-580 cells compared to parental wild-type (WT) cells. The effluxes of Rh-123 and Dau were ATP- and temperature-dependent. The p-glycoprotein inhibitors, verapamil, cyclosporin A and reserpine blocked the efflux of both Rh-123 and Dau. In contrast, the inhibitors of MRP, MK571, BSO-treatment, arsenite and genistein did not block the efflux of either Rh-123 or Dau from MQ-580 cells. These findings suggest that the p-glycoprotein is the major transporter involved in effluxing Rh-123 and Dau from MQ-580 cells.

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.

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.

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.