RESUMO
Following DNA damage, cells undergo G2/M cell cycle arrest, allowing time for DNA repair. G2/M checkpoint activation involves activation of Wee1 and Chk1 kinases and inhibition of Cdc25A and Cdc25C phosphatases, which results in inhibition of Cdc2 kinase. Results presented in this report indicate that gamma-irradiation (IR) exposure of MCF-7 cells resulted in extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation and induction of G2/M arrest. Furthermore, inhibition of ERK1/2 signaling resulted in >or=85% attenuation in IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ataxia telangiectasia mutated- and rad3-related (ATR), Chk1 and Wee1 kinases as well as phosphorylation of Cdc25A-Thr506, Cdc25C-Ser216 and Cdc2-Tyr15. Moreover, incubation of cells with caffeine, which inhibits ataxia telangiectasia mutated (ATM)/ATR, or transfection of cells with short interfering RNA targeting ATR abrogated IR-induced Chk1 phosphorylation and G2/M arrest but had no effect on IR-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling resulted in marked attenuation in IR-induced ATR activity with little, if any, effect on IR-induced ATM activation. These results implicate IR-induced ERK1/2 activation as an important regulator of G2/M checkpoint response to IR in MCF-7 cells.
Assuntos
Divisão Celular/efeitos da radiação , Dano ao DNA , Fase G2/efeitos da radiação , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Fosfatases cdc25/metabolismoRESUMO
A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels of hFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of [3H]methotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity.
Assuntos
Proteínas de Transporte/genética , Resistência a Medicamentos/fisiologia , Antagonistas do Ácido Fólico/farmacologia , Metotrexato/farmacologia , Animais , Células CHO/fisiologia , Cricetinae , DNA Recombinante/genética , Receptores de Folato com Âncoras de GPI , Ácido Fólico/metabolismo , Humanos , Immunoblotting , Células KB/ultraestrutura , RNA/análise , Receptores de Superfície Celular/genética , Transfecção , Células Tumorais Cultivadas/fisiologiaRESUMO
Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene-amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene-transfected cells contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFR gene sequences responsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected.
Assuntos
Genes , Tetra-Hidrofolato Desidrogenase/genética , 5'-Nucleotidase , Animais , Sequência de Bases , Sangue , Neoplasias da Mama , Linhagem Celular , Clonagem Molecular , Meios de Cultura , DNA/metabolismo , Replicação do DNA , Feminino , Amplificação de Genes , Humanos , Mutação , NucleotidasesRESUMO
The ability of malignant cells to develop resistance to cytotoxic drugs poses a major obstacle to the ultimate success of cancer therapy. While some mechanisms of resistance allow cells to survive exposure to a single agent, the phenomenon of multidrug resistance (MDR) confers upon cells the ability to withstand exposure to lethal doses of many structurally unrelated antineoplastic agents. MDR has been strongly linked to the overexpression of a membrane-associated glycoprotein, P-glycoprotein, which appears to play a role in drug efflux. However, several lines of evidence suggest that other mechanisms of resistance are involved in MDR; biochemical similarities observed in a human breast cancer cell line after the acquisition of MDR and in carcinogen-induced rat preneoplastic hepatic nodules indicate that changes in regulation of phase I and phase II drug-metabolizing enzymes may also play a role in MDR. An atypical pattern of MDR has been characterized and related to altered topoisomerase activity. Improvement in current cancer chemotherapy may be achieved by interfering with the regulation and expression of mechanisms of MDR.
Assuntos
Antineoplásicos/uso terapêutico , Resistência a Medicamentos/genética , Neoplasias Experimentais/tratamento farmacológico , Neoplasias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias Experimentais/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
We have studied the effects of estrogen and the antiestrogen tamoxifen on the regulation of dihydrofolate reductase (DHFR) gene expression in a methotrexate-resistant (MTXR) human breast cancer cell line MCF-7, which contains a 50-fold increase in the level of DHFR enzyme and amplified DHFR gene sequences. Despite their selection for methotrexate resistance, the MTXR cells have retained many characteristics of the parental MCF-7 cell line. Concentrations of estrogen receptors as well as their binding affinity to estradiol are identical in both cell lines. MTXR MCF-7 cells remain sensitive to estrogen and respond to estradiol with an induction of progesterone receptors, as well as increases in the rate of DNA synthesis and cell growth. Incubation of MTXR MCF-7 cells with estradiol results in an additional 1.5- to 3.0-fold increase in their already elevated level of DHFR. The hormone-induced increases in DNA synthesis and DHFR levels are similar both with respect to the time course of inductions, as well as their dose response to estradiol. However, these two estrogen-induced effects are not coupled, since the induction of DHFR occurs even in the absence of concomitant DNA synthesis. Estradiol has no effect on DHFR enzyme stability; thus, the entire effect of estrogen on DHFR levels results from the increased synthesis of this housekeeping enzyme. In contrast, treatment of MTXR MCF-7 cells with the antiestrogen tamoxifen reduces the rate of DHFR enzyme synthesis, resulting in lower cellular levels of DHFR. These MTXR MCF-7 cells represent a useful model in which to study the mechanisms involved in the modulation of DHFR gene expression by estrogen and tamoxifen. Since the level of DHFR is a critical determinant of methotrexate cytotoxicity understanding, the regulation of DHFR gene expression may have clinical implications for the use of hormonal therapy in combination with chemotherapy for the treatment of breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Tamoxifeno/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular , Linhagem Celular , DNA de Neoplasias/biossíntese , Resistência a Medicamentos , Feminino , Humanos , Metotrexato/farmacologiaRESUMO
We have previously described a mitoxantrone-resistant human breast carcinoma cell line, MCF7/MX, in which resistance was associated with a defect in the energy-dependent accumulation of mitoxantrone in the absence of P-glycoprotein overexpression (M. Nakagawa et al., Cancer Res. 52: 6175-6181, 1992). We now report that this cell line is highly cross-resistant to the camptothecin analogues topotecan (180-fold), 9-aminocamptothecin (120-fold), CPT-11 (56-fold), and SN38 (101-fold), but is only mildly cross-resistant to the parent compound camptothecin (3.2-fold) and 10,11-methylenedioxy-camptothecin (2.9-fold). Topotecan accumulation was decreased in MCF7/MX cells compared to parental MCF7/WT cells, and there was a corresponding reduction in topotecan-mediated stimulation of the enzyme/DNA complex formation in MCF7/MX cells compared to MCF7/WT cells. No overexpression of the multidrug resistance-associated protein was detected compared to parental MCF7/WT cells. Furthermore, both sensitive MCF7/WT and mitoxantrone-resistant MCF7/MX cells contain equal amounts of DNA topoisomerase I protein, and DNA relaxation activities were equal in both cell lines and inhibited to the same extent by topotecan and camptothecin. Thus, these results suggest a novel mechanism of resistance to topoisomerase I inhibitors in cancer cells.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Mitoxantrona/farmacologia , Inibidores da Topoisomerase I , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Camptotecina/metabolismo , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Resistência a Medicamentos , Feminino , Humanos , Topotecan , Células Tumorais CultivadasRESUMO
BRCA1 mRNA and protein levels are regulated by the steroid hormones estrogen and progesterone in human breast cancer cells. BRCA1 mRNA and protein levels were significantly decreased in estrogen-depleted MCF-7 and BT20T cells and increased again after stimulation with beta-estradiol. The increase in BRCA1 expression upon stimulation with estrogen was not coordinated with the early induction of the estrogen-dependent pS2 gene but closely paralleled the delayed increase in the S-phase dependent marker cyclin A. T47-D cells deprived of steroid hormones and subsequently stimulated with progesterone also showed a delayed increase in BRCA1 mRNA expression. However, no change in BRCA1 protein was detected in these cells. When considered together, the data suggest that steroid hormones may affect BRCA1 expression indirectly by altering the proliferative status of the cells rather than acting directly on DNA sequences in the BRCA1 gene itself.
Assuntos
Neoplasias da Mama/genética , Estradiol/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas , Fatores de Transcrição/metabolismo , Proteína BRCA1 , Ciclinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Progesterona/farmacologia , RNA Neoplásico/genética , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Fator Trefoil-1 , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug. MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine. Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells. This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process. Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells. Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro. However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT. Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells. Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.
Assuntos
Neoplasias da Mama/metabolismo , Glicoproteínas de Membrana/fisiologia , Mitoxantrona/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Anticorpos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Reações Cruzadas , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/farmacologia , Resistência a Medicamentos , Etoposídeo/farmacologia , Expressão Gênica/genética , Humanos , Líquido Intracelular/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitoxantrona/farmacologia , Células Tumorais Cultivadas , Verapamil/farmacologiaRESUMO
We found that Adriamycin increased the pentose phosphate shunt activity in both Adriamycin-sensitive (WT) and Adriamycin-resistant (ADRR) human breast cancer MCF-7 cells. In contrast, hydrogen peroxide and cumene hydroperoxide markedly stimulated pentose-shunt activity in ADRR but only moderately increased the activity in WT cells. Furthermore, the altered oxidation-reduction regulation is associated with changes intrinsic to the key enzymes of the pentose-shunt pathway, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase and with glutathione peroxidase. We found the Vmax values for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were 50- and 4-fold lower, respectively, in ADRR than WT cells and the Kms of NADP+ were 10-fold lower in ADRR than WT. The activity of glutathione reductase in ADRR is 42% of that in WT. In spite of these changes, the response of the cells to both hydrogen peroxide and organic peroxide is not limited by either the capacity of the pentose shunt or glutathione reductase, but is determined by the activity of glutathione peroxidase and a glutathione transferase which possess peroxidase activity. The kinetic properties of the glucose-6-phosphate dehydrogenase in ADRR may, however, seriously limit the activity of cytochrome P-450 reductase, a major enzyme of Adriamycin conversion to a free radical.
Assuntos
Derivados de Benzeno/farmacologia , Doxorrubicina/farmacologia , Peróxido de Hidrogênio/farmacologia , Via de Pentose Fosfato/efeitos dos fármacos , Neoplasias da Mama , Linhagem Celular , Resistência a Medicamentos , Feminino , Humanos , Cinética , Azul de Metileno/farmacologiaRESUMO
Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.
Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Glutationa Transferase/genética , Transfecção , Sequência de Bases , Neoplasias da Mama , Linhagem Celular , Células Clonais , Feminino , Biblioteca Gênica , Vetores Genéticos , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Humanos , Cinética , Fígado/enzimologia , Mapeamento por RestriçãoRESUMO
A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity.
Assuntos
Neoplasias da Mama/enzimologia , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo/metabolismo , Teniposídeo/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Proteínas de Transporte/análise , DNA Topoisomerases Tipo I/análise , DNA Topoisomerases Tipo II/análise , DNA Topoisomerases Tipo II/genética , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Etoposídeo/farmacologia , Feminino , Humanos , Glicoproteínas de Membrana/análise , RNA Mensageiro/análise , Teniposídeo/farmacologia , Células Tumorais CultivadasRESUMO
p27Kip1, a cyclin-dependent kinase inhibitor, is recognized as a negative regulator of the cell cycle. In this paper, we report that overexpression of p27Kip1 triggers apoptosis in several different human cancer cell lines. Using a recombinant adenoviral vector that expresses p27Kip1 (Adp27), we found that overexpression of p27Kip1 in MDA-MB-231 breast cancer cells induces apoptosis that was seen by a number of different techniques, including flow cytometry and in situ terminal deoxynucleotidyl transferase-mediated nick end labeling, flow cytometric assay for sub-G1 population, and 4',6-diamindino-2-phenylindole staining. Cleavage of poly(ADP-ribose) polymerase and degradation of cyclin B1, events that are known to be associated with apoptosis, were also observed following overexpression of p27Kip1. This is the first report indicating a role for p27Kip1 in induction of apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Supressoras de Tumor , Adenoviridae/genética , Adenoviridae/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Inibidor de Quinase Dependente de Ciclina p27 , Feminino , Vetores Genéticos , Humanos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Células Tumorais CultivadasRESUMO
An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (AdrR) cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins. Cytogenetic analysis of the AdrR cell line showed the presence of homogeneously staining regions on several chromosomes which were not present in the parental cell line. Using the technique of in-gel renaturation, DNA sequences which were amplified 50- to 100-fold in the AdrR cell line and which covered a total of over 140 kilobases were isolated. In addition, AdrR cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences. A hamster cDNA P-glycoprotein gene probe was used to screen a lambda gt10 cDNA library made from human AdrR cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybridization studies with the cloned human cDNA (pADR1) showed that the AdrR MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the AdrR cell line contained a 4.8-kilobase transcript which was overexpressed 45-fold. There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycin-resistant cell lines. In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the AdrR MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the AdrR cells.
Assuntos
Neoplasias da Mama/genética , DNA de Neoplasias/análise , DNA/análise , Doxorrubicina/farmacologia , Amplificação de Genes , Sequência de Bases , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular , Resistência a Medicamentos , Feminino , Glicoproteínas/genética , Humanos , RNA/análise , Vincristina/farmacologiaRESUMO
Our laboratory has previously reported the isolation of a murine cDNA which restores reduced folate carrier (RFC) activity and methotrexate (MTX) sensitivity to a MTX-resistant, transport-deficient human breast cancer cell line (MTXR ZR-75-1) (K. H. Dixon et al., J. Biol. Chem., 269: 17-20, 1994). Using this murine cDNA as a probe, we have isolated two homologous overlapping partial cDNAs from a human testis cDNA library. In addition, using human cDNA as a probe, we have isolated a 20-kb human genomic fragment which contains RFC coding regions. Analysis of the nucleotide sequence of these clones revealed that the human RFC gene, RFC1, is approximately 65% homologous to the murine and hamster genes. Using a human genomic P1 plasmid clone containing RFC1, we mapped the location of RFC1 by fluorescence in situ hybridization to the end of the long arm of chromosome 21 (21q22.2-q22.3). Fluorescence in situ hybridization analysis also showed that two copies of RFC1 were present in MTXR ZR-75-1 cells, and showed no evidence of rearrangement of this gene. Northern blot analysis of MTXR ZR-75-1 cells demonstrated a marked decrease in the level of the 3-kb RFC1 transcript relative to the parental cell line, and Western blot analysis using a polyclonal antibody raised against a peptide generated from the RFC1 sequence showed decreased expression of an approximately M(r) 56,000 protein in MTXR ZR-75-1 cells. Finally, MTXR ZR-75-1 cells transfected with an RFC1 gene showed increased MTX uptake, which was more sensitive to competition by folinic acid than by folic acid. Therefore, decreased RFC1 expression appears to be the molecular mechanism of decreased MTX uptake in this MTX-resistant cell line.
Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Ácido Fólico/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama/metabolismo , Proteínas de Transporte/química , Resistência a Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Proteínas de Membrana/química , Metotrexato , Dados de Sequência Molecular , Peso Molecular , Células Tumorais CultivadasRESUMO
31P magnetic resonance spectra of perfused human breast cancer cells with the phenotype of pleiotropic drug resistance exhibit striking differences in the levels of phosphate metabolites from the wild-type, drug-sensitive parent cell line. Resistant cells demonstrated elevated levels of phosphocreatine and depressed levels of phosphomonoesters, phosphodiesters, and diphosphodiesters. These differences may reflect significant alterations in the control of bioenergetic metabolism between drug-resistant and -sensitive cells.
Assuntos
Neoplasias da Mama/metabolismo , Espectroscopia de Ressonância Magnética , Fosfatos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular , Resistência a Medicamentos , Metabolismo Energético , Feminino , Humanos , Concentração de Íons de HidrogênioRESUMO
A Phase I clinical trial of 1-beta-D-arabinofuranosyl-5-azacytosine (ara-AC or fazarabine) given as a 72-h continuous infusion on a 21-day cycle was conducted in 27 adult patients with refractory cancer. The major toxicity was reversible granulocytopenia and thrombocytopenia. Dose-limiting toxicity was observed at a dose rate of 1.96 mg/m2/h in which Grade IV leukopenia (WBC less than 1,000/mm3) occurred in 4 of 11 patients and Grade IV thrombocytopenia (platelets less than 25,000/mm3) occurred in 3 of 11 patients. Plasma steady-state levels ranged from 0.13 to 0.6 microM for doses of 1.25 to 5.94 mg/m2/h. Mean total body clearance was 647 ml/min/m2. Minor clinical responses were seen in one patient with testicular cancer, one patient with colon cancer, one patient with breast cancer, and one patient with acute nonlymphocytic leukemia. Another patient with adenocarcinoma of unknown primary had stable disease during 13 cycles of therapy. Based on the results of this study, the recommended dose for Phase II studies of 1-beta-D-arabinofuranosyl-5-azacytosine administered as a 72-h continuous infusion is 2.0 mg/m2/h (48 mg/m2/day).
Assuntos
Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Neoplasias/tratamento farmacológico , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Azacitidina/administração & dosagem , Azacitidina/efeitos adversos , Azacitidina/farmacocinética , Ensaios Clínicos como Assunto , Esquema de Medicação , Avaliação de Medicamentos , Feminino , Humanos , Leucopenia/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neutropenia/induzido quimicamente , Trombocitopenia/induzido quimicamenteRESUMO
Piroxantrone is an anthrapyrazole derivative with broad antitumor activity in vitro. In previous phase I trials, the dose-limiting toxicity of this agent was myelosuppression. Therefore, a phase I and pharmacokinetic study of a 1-h infusion of piroxantrone in combination with granulocyte-colony stimulating factor was conducted. In this article, we report the results of the pharmacokinetic analysis. Thirty-seven patients were studied over a dosage range of 150 to 555 mg/m2. The plasma elimination of piroxantrone was biexponential with a mean (+/- SD) t1/2 alpha of 3.2 +/- 2.7 min and a mean (+/- SD) t1/2 beta of 82 +/- 92 min. Clearance was 840 +/- 230 ml/min/m2. A limited sampling strategy was developed to allow the estimation of total drug exposure (area under the plasma concentration-time curve) from the plasma piroxantrone concentrations at 30, 60, and 120 min after the start of the infusion. The pharmacokinetic behavior of a presumed piroxantrone metabolite not previously described in plasma was also characterized. Based on in vitro cytotoxicity studies with partially purified extract of this compound, we do not believe that it contributes to the antitumor effects of piroxantrone at the concentrations observed in plasma. Finally, piroxantrone elimination was linear over the nearly 4-fold dose range studied, indicating that when dose adjustments are made, systemic drug exposure will remain predictable.
Assuntos
Antraquinonas/farmacocinética , Antineoplásicos/farmacocinética , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Neoplasias/metabolismo , Pirazóis/farmacocinética , Adulto , Antraquinonas/administração & dosagem , Antraquinonas/sangue , Antraquinonas/urina , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Antineoplásicos/urina , Humanos , Neoplasias/tratamento farmacológico , Pirazóis/administração & dosagem , Pirazóis/sangue , Pirazóis/urinaRESUMO
The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells. The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues. The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.
Assuntos
Glutationa Transferase/genética , Isoenzimas/genética , Glicoproteínas de Membrana/genética , Neoplasias/análise , RNA/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Sequência de Bases , DNA/análise , Resistência a Medicamentos , Regulação da Expressão Gênica , Humanos , Dados de Sequência MolecularRESUMO
In this study we examine the relationship between p21CIP1/Waf1 (CIP1), a 21 kDa protein that binds to and modulates the activity of several cyclin dependent kinases and expression of wild-type (WT) p53 in human breast epithelial cells. Basal CIP1 protein, but not CIP1 mRNA levels correlated well with expression of WT p53 in human breast epithelial cells. To obtain more direct evidence that WT p53 regulated the level of CIP1 protein, the Human Papilloma Virus (HPV) E6 protein was introduced into immortalized 184B5 breast cells. Residual WT p53 levels correlated well with CIP1 protein but not CIP1 mRNA levels in isolated clones of transfected cells. CIP1 protein was increased at early times after growth factor arrested cells were stimulated to proliferate. The rise in CIP1 protein was due to a concomitant increase in CIP1 mRNA levels in MCF10, but not in normal mammary epithelial cells. DNA damage induced by ionizing radiation resulted in a transient increase in WT p53 levels but a prolonged induction of CIP1 protein. The sustained increase in CIP1 protein 24 h after radiation could not be attributed to a concomitant increase in CIP1 mRNA levels. Although the half-life of the CIP1 protein was not altered following irradiation, a fourfold increase in the amount of radioactivity incorporated into CIP1 protein was detected. When considered together these data suggest that wild-type p53 affects CIP1 protein accumulation at a posttranscriptional level in human breast epithelial cells under different physiologic and stress conditions.
Assuntos
Mama/fisiologia , Ciclo Celular , Ciclinas/genética , Dano ao DNA , Genes p53 , Mama/ultraestrutura , Ciclo Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Células Epiteliais , Epitélio/fisiologia , Expressão Gênica , Humanos , RNA Mensageiro/fisiologia , Transcrição GênicaRESUMO
rhoA encodes a ras-related GTP-binding protein that is thought to play a role in cytoskeletal organization. Recent evidence has suggested both that rhoA could act either as a dominant oncogene, since transfection of both normal and activated rho genes confer a transformed phenotype on fibroblast cells in culture, or as a recessive tumor suppressor gene, by virtue, in part, of its chromosomal location at 3p21, a site deleted in many human malignancies. In either case, a role for rhoA in the oncogenesis of human tumors would be supported by the finding of rhoA mutations in tumors. We therefore examined human tumors and cell lines for mutations in the protein coding regions of rhoA by RNAase protection analysis. We first examined the expression of rhoA in renal cell carcinoma cell lines in which 3p21 was heterozygously deleted or retained. We found no evidence for rhoA mutations in these specimens. We also examined RNA from lung, breast, colon or ovarian tumors and also found no evidence of activating rhoA mutations. Furthermore, there was no relation between the level of rhoA mRNA expression and the presence or absence of 3p21 deletions in the renal cell carcinoma specimens. Thus, although rhoA has transforming potential in vitro, there is no evidence that it is activated by mutation in human malignancies, or that it could act as a tumor suppressor gene in tumors in which 3p21 is deleted.