Effect of P-glycoprotein on flavopiridol sensitivity.

Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC(50)s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CH(R)C5 (higher Pgp) cells were 90.2 +/- 6.6 nM and 117 +/- 2.3 nM, respectively (P< 0.01), suggesting that Pgp might have a modest effect on flavopiridol action. Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CH(R)C5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp.

The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux.

Because the activities of HER family members are elevated and/or aberrant in a variety of human neoplasms, these cell surface receptors are receiving increasing attention as potential therapeutic targets. In the present study, we examined the effect of combining the HER family tyrosine kinase inhibitor CI1033 (PD 183805) with the topoisomerase (topo) I poison 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in a number of different cell lines. Colony-forming assays revealed that the antiproliferative effects of simultaneous treatment with CI1033 and SN-38 were synergistic in T98G glioblastoma cells and HCT8 colorectal carcinoma cells, whereas sequential treatments were additive at best. In additional studies examining the mechanistic basis for these findings in T98G cells, immunoblotting revealed that the inhibitory effects of CI1033 on epidermal growth factor receptor autophosphorylation were unaffected by SN-38. Likewise, CI1033 had no effect on topo I polypeptide levels, localization, or activity. Nonetheless, CI1033 markedly enhanced the number of covalent topo I-DNA complexes stabilized by SN-38 or the related agent topotecan (TPT). Analysis of intracellular SN-38 levels by high-performance liquid chromatography and intracellular TPT levels by flow microfluorometry revealed that CI1033 increased the steady-state accumulation of SN-38 and TPT by 9.4 +/- 1.9- and 1.8 +/- 0.2-fold, respectively. Further evaluation revealed that the initial rate of TPT uptake was unaffected by CI1033, whereas the rate of efflux was markedly diminished. Additional studies demonstrated that T98G and HCT8 cells express the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. Moreover, CI1033 enhanced the uptake and cytotoxicity of SN-38 and TPT in cells transfected with BCRP but not empty vector. Conversely, CI1033 accumulation was diminished in cells expressing BCRP, suggesting that CI1033 is a substrate for this efflux pump. These results indicate that CI1033 can modulate the accumulation and subsequent cytotoxicity of two widely used topo I poisons in cells that have no history of previous exposure to these agents.

Characterization of an ovarian carcinoma cell line resistant to cisplatin and flavopiridol.

Flavopiridol, the first inhibitor of cyclin-dependent kinases to enter clinical trials, has shown promising antineoplastic activity and is currently undergoing Phase II testing. Little is known about mechanisms of resistance to this agent. In the present study, we have characterized an ovarian carcinoma cell line [OV202 high passage (hp)] that spontaneously developed drug resistance upon prolonged passage in tissue culture. Standard cytogenetic analysis and spectral karyotyping revealed that OV202 hp and the parental low passage line OV202 shared several marker chromosomes, confirming the relatedness of these cell lines. Immunoblotting demonstrated that OV202 and OV202 hp contained similar levels of a variety of polypeptides involved in cell cycle regulation, including cyclin-dependent kinases 2 and 4; cyclins A, D1, and E; and proliferating cell nuclear antigen. Despite these similarities, OV202 hp was resistant to flavopiridol and cisplatin, with increases of 5- and 3-fold, respectively, in the mean drug concentrations required to inhibit colony formation by 90%. In contrast, OV202 hp and OV202 displayed indistinguishable sensitivities to oxaliplatin, paclitaxel, topotecan, 1,3-bis(2-chloroethyl)-1-nitrosourea, etoposide, doxorubicin, vincristine, and 5-fluorouracil, suggesting that the spontaneously acquired resistance was not attributable to altered P-glycoprotein levels or a general failure to engage the cell death machinery. After incubation with cisplatin, whole cell platinum and platinum-DNA adducts measured using mass spectrometry were lower in OV202 hp cells than OV202 cells. Similarly, after flavopiridol exposure, whole cell flavopiridol concentrations measured by a newly developed high performance liquid chromatography assay were lower in OV202 hp cells. These data are consistent with the hypothesis that acquisition of spontaneous resistance to flavopiridol and cisplatin in OV202 hp cells is due, at least in part, to reduced accumulation of the respective drugs. These observations not only provide the first characterization of a flavopiridol-resistant cell line but also raise the possibility that alterations in drug accumulation might be important in determining sensitivity to this agent.

Effect of 6-aminonicotinamide and other protein synthesis inhibitors on formation of platinum-DNA adducts and cisplatin sensitivity.

The present study was undertaken to examine the mechanistic basis for the recent observation that the pyridine nucleotide derivative 6-aminonicotinamide (6AN, NSC 21206) enhances the accumulation and resulting cytotoxicity of cisplatin in a variety of tumor cell lines. When A549 lung cancer cells or K562 leukemia cells were treated with 62.5 microM 6AN for 21 h and then pulse-labeled with [(35)S]methionine for 1 h, increased labeling of five polypeptides, one of which corresponded to a M(r) approximately 78,000 glucose-regulated protein (GRP78), was observed. Two subsequent observations, however, suggested that up-regulation of these polypeptides was unlikely to explain the interaction between 6AN and cisplatin: 1) the concentration of 6AN required to induce GRP78 was 4-fold higher than the dose required to sensitize cells to cisplatin; and 2) simultaneous treatment of cells with 6AN and cycloheximide prevented the increase in GRP78 but not the sensitizing effect of 6AN. On the contrary, treatment with the protein synthesis inhibitors cycloheximide, anisomycin, or puromycin as well as prolonged exposure to the RNA synthesis inhibitor actinomycin D mimicked the biochemical modulating effects of 6AN on cisplatin action. Conversely, 6AN inhibited protein synthesis, whereas 18 6AN analogs that failed to enhance Pt-DNA adducts and cisplatin cytotoxicity failed to inhibit protein synthesis. These observations are consistent with a model in which 6AN and other inhibitors of protein synthesis act as modulating agents by increasing cisplatin accumulation, thereby enhancing the formation of Pt-DNA adducts and subsequent cisplatin-induced cell death.

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent.

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

A one-step method for protein estimation in biological samples: nitration of tyrosine in nitric acid.

A number of methods are commonly employed for the determination of protein in biological samples. Unfortunately, several compounds that are constituents of biological buffers interfere with these methods, limiting their application. Previous studies have demonstrated that tyrosine rapidly undergoes nitration in nitric acid to yield 3-nitrotyrosine, which has a lambdamax of 358 nm. Utilizing this reaction, we have developed a one-step method for the assessment of protein content in biological samples. Common interfering substances, including SDS, urea, glycerol, ammonium sulfate, and beta-mercaptoethanol, do not interfere with this method. Because of its simplicity, this reaction might be useful for estimating protein content in a variety of biological samples.