Modulation of activity of the promoter of the human MDR1 gene by Ras and p53.

Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.

The promyelocytic leukemia protein interacts with Sp1 and inhibits its transactivation of the epidermal growth factor receptor promoter.

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth- and transformation-suppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-bp element (the sequences between -150 and -16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST-Sp1 proteins and in vitro-translated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia.

The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.

Interaction of P-glycoprotein with protein kinase C in human multidrug resistant carcinoma cells.

Indirect evidence has suggested that P-glycoprotein (P-gp), the multidrug transporter, is phosphorylated by protein kinase C (PKC) and that phosphorylation modulates its transport function. To address the first premise more directly, ie., that P-gp is phosphorylated by PKC, we investigated the interaction between P-gp and PKC in sensitive and multidrug resistant MCF-7 and KB human carcinoma cell lines. We found that P-gp and PKC were coimmunoprecipitated from the multidrug-resistant cell lines MCF-7/AdrR and KB-V-1, using antibodies to either protein. The association between the two proteins was enhanced by phorbol 12-myristate 13-acetate, an analogue of diacylglycerol that induces translocation of PKC to the plasma membrane. The anti-P-gp immunoprecipitates contained PKC activity as measured by direct phosphorylation reactions. The interaction of PKC with P-gp displayed isozyme specificity: PKC-alpha, -beta, gamma, -epsilon, and -phi, but not -delta, -mu, -zeta, -lambda, were found to coimmunoprecipitate with P-gp. These studies indicate that P-gp closely interacts with PKC and serves as a substrate, and that specific isozymes of this kinase may be involved in the phosphorylation of the multidrug transporter.

Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cells by cDNA microarray.

Drug resistance in cancer is a major obstacle to successful chemotherapy. Cancer cells exposed to antitumor drugs may be directly induced to express a subset of genes that could confer resistance, thus allowing some cells to escape killing and form the relapsed resistant tumor. Alternatively, some cancer cells may be expressing an array of genes that could confer intrinsic resistance, and exposure to cytotoxic drugs select for the survival of these cells that form the relapsed tumor. We have used cDNA microarray to monitor the expression profiles of MCF-7 cells that are either transiently treated with doxorubicin or selected for resistance to doxorubicin. Our results showed that transient treatment with doxorubicin altered the expression of a diverse group of genes in a time-dependent manner. A subset of the induced genes was also found to be constitutively overexpressed in cells selected for resistance to doxorubicin. This distinct set of overlapping genes may represent the signature profile of doxorubicin-induced gene expression and resistance in cancer cells. Our studies demonstrate the feasibility of obtaining potential molecular profile or fingerprint of anticancer drugs in cancer cells by cDNA microarray, which might yield further insights into the mechanisms of drug resistance and suggest alternative methods of treatment.

Gene-specific DNA repair and steady state transcription of the MDR1 gene in human tumor cell lines.

We have explored the relationship between DNA repair and transcription in vivo. A gene-specific repair assay has been employed to study removal of ultraviolet light-induced cyclobutane pyrimidine dimers in the MDR1 gene at different levels of MDR1 mRNA expression. The parental human adenocarcinoma cell line, KB-3-1, has very low levels of MDR1 mRNA expression, but its multidrug resistant derivatives KB-8-5 and KB-C1 have 42-fold and 3800-fold increases in MDR1 mRNA expression, respectively. In the KB-3-1 cell line that has a low level of MDR1 mRNA expression, we find a low level of MDR1 gene-specific repair and inefficient repair of the transcribed strand of the gene. In the KB-8-5 cell line that has a modest increase in MDR1 mRNA expression, we find only a minor increase in dimer repair in the MDR1 gene. Here, the repair in the transcribed strand is not significantly higher than that in the KB-3-1 cell line. However, in the KB-C1 derivative, where there is a 3800-fold increase in the level of MDR1 mRNA expression, we find a substantial increase in the level of dimer repair in the MDR1 gene. In addition, the MDR1 transcribed strand repair is markedly more efficient than the repair in the nontranscribed strand. Our data suggest that the rate of transcription in the MDR1 gene must be substantially increased before there is any measurable effect on DNA repair. Repair in the housekeeping gene, dihydrofolate reductase (DHFR), was similar in all three tumor cell lines. Repair in its transcribed strand was markedly lower than previously reported in normal human fibroblasts. We suspect that these human HeLa-derived tumor cell lines have deficient gene-specific DNA repair. This may be an important aspect of their malignant phenotype.

Function and regulation of the human multidrug resistance gene.

The discovery of an energy-dependent pump system for natural product anticancer drugs has important implications for the biology of related energy-dependent transport systems as well as for the treatment of human cancer. To fully realize the therapeutic potential associated with manipulation of the multidrug transporter, it will be necessary to understand the mechanisms of action of the transporter and its mode of regulation. This review has summarized recent developments in these areas which suggest that both the activity of the pump and its genetic regulation are potential targets for new anticancer therapies.

Cisplatin resistance in cyclic AMP-dependent protein kinase mutants.

The emergence of cisplatin resistance poses a major problem to the successful treatment of a variety of human malignancies. Therefore, understanding the molecular mechanisms that underlie cisplatin resistance could significantly improve the clinical efficacy of this cytotoxic agent. Various studies have described that cellular sensitivity to cisplatin can be influenced by several signal transduction pathways. In this review, we examine the role of the cyclic AMP-dependent protein kinase (PKA) in the modulation of drug resistance in cancer. By a somatic mutant genetic approach, the role of PKA in the development of resistance to chemotherapeutic agents has been investigated. A series of mutants with decreased PKA activity was examined for their sensitivity to cisplatin. PKA mutants with defective regulatory (RIalpha) subunits, but not altered catalytic (C) subunits, exhibit increased resistance to cisplatin, as well as other DNA-damaging agents. Furthermore, since RIalpha subunit mutants show enhanced DNA repair we, therefore, hypothesize that functional inactivation of PKA may result in increased recognition and repair of cisplatin lesions. Alternatively, it seems likely that mutation of the RIalpha subunit may affect cellular sensitivity to various anticancer drugs, suggesting that the RIalpha subunit may have other physiological functions in addition to inhibiting the kinase activity of the C subunit. Therefore, exploitation of cyclic AMP levels or functional alteration of the R subunit may potentiate the cytotoxicity of chemotherapeutic agents and circumvent drug resistance in cancer. More importantly, the altered pattern and mechanism of drug resistance may offer the opportunity to investigate novel regulatory functions of the RIalpha subunit of PKA.

Extracellular catalytic subunit activity of the cAMP-dependent protein kinase in prostate cancer.

The role of cAMP in cell growth and differentiation, gene expression, and neuronal function is mediated by the cAMP-dependent protein kinase (PKA). Differential expression of type I and type II PKA has been correlated with neoplastic transformation and differentiation, respectively. PKA is primarily an intracellular enzyme. However, it has been demonstrated that PKA may be associated with the plasma membrane and is exposed to the extracellular environment. Here we report the first evidence for the presence of a free extracellular kinase activity of PKA in the growth media of cultured prostate and other cancer cells, as well as in plasma samples from prostate cancer patients. This PKA activity is specific due to its phosphorylation of the PKA-specific substrate kemptide and its inhibition by the potent and specific PKA inhibitor PKI, but not by other protein kinase-inhibitory peptides. Intriguingly, this exoprotein kinase activity is cAMP independent, suggesting that only the catalytic subunit is secreted, and therefore the kinase activity is not modulated by the regulatory subunit of PKA. Western blot analysis of the culture supernatant from prostate cancer cells indicates the presence of the catalytic subunit. This increase in extracellular PKA catalytic subunit activity in prostate cancer may have profound effects on the tumorigenesis of prostate cancer and may serve as a novel marker and therapeutic target for the disease.

Role of cAMP-dependent protein kinase in the regulation of DNA repair.

Enhanced DNA repair is an important factor in drug resistance in cancer. Using cell-free extracts derived from the fission yeast, Schizosaccharomyces pombe, we demonstrate in an in vitro system DNA repair system that increased cAMP levels, which activates cAMP-dependent protein kinase (PKA), inhibits repair of ultraviolet (UV)-damaged DNA. Supplementing the cell-free system with the catalytic kinase subunit of PKA also inhibits DNA repair. In contrast, addition of the PKA inhibitor H-89 enhances repair activity. These results show that PKA regulates DNA repair synthesis, thus implicating the cAMP signaling pathway in DNA damage response and repair of UV-damaged DNA lesions.

Nonradioactive detection of DNA repair synthesis.

DNA repair is essential for the surveillance and maintenance of the integrity of the genome in response to various insults that damage DNA. The development of cell-free repair systems using radiolabeled nucleotide to monitor repair synthesis of exogenously introduced damaged-plasmid DNA has enabled the analysis of specific proteins required for repair synthesis. However, the hazards and the burgeoning cost of using radioisotopes have become significant factors in the laboratory. We describe here the use of digoxigenin-dUTP in place of radioactivity in a nonradioactive cell-free repair assay to detect DNA repair.

Transactivation of the human multidrug resistance (MDR1) gene promoter by p53 mutants.

Multidrug resistance in human cancer is associated with overexpression of the MDR1 gene, which encodes a plasma membrane energy-dependent efflux pump termed P-glycoprotein (or the multidrug transporter), which confers cross-resistance to multiple hydrophobic natural product cytotoxic drugs. We have previously shown in cotransfection experiments that activity of the human MDR1 gene promoter is modulated by Ras and p53, suggesting that expression of the MDR1 gene may be associated with the activation of oncogenes and/or functional loss of tumor suppressor genes during oncogenesis. To further characterize the effects of p53 on the MDR1 promoter, we have shown in the current study that the region of the promoter that is required for transactivation by p53 mutants overlaps with the region that is essential for basal promoter activity. In addition, we also have shown that several different p53 mutants transactivate the MDR1 promoter in several different cell types, including embryo fibroblasts derived from the p53-deficient (p53-l-) mice generated by gene targeting.

Cisplatin sensitivity in cAMP-dependent protein kinase mutants of Saccharomyces cerevisiae.

The emergence of cisplatin resistance poses a significant problem to the treatment of a variety of human malignancies. Therefore, understanding the molecular basis of cisplatin resistance could improve the clinical effectiveness of this anticancer agent. Recently, our laboratory has demonstrated that cAMP-dependent protein kinase (PKA) mutants of the Chinese hamster ovary (CHO) and the mouse adrenocortical carcinoma Y1 cells exhibited increased resistance to cisplatin as well as other DNA-damaging drugs. Further studies showed that either the functional inactivation of PKA or the mutation in the regulatory subunit gene may cause increased recognition of cisplatin-damaged DNA and enhanced DNA repair capacity. In this study, we evaluated the role of PKA in modulating cellular sensitivity to cisplatin in a series of PKA mutants of Saccharomyces cerevisiae. Mutants with decreased kinase activity resulting from a srv2 mutation showed no alterations in cisplatin sensitivity. Complementation of TPK1 in a yeast strain containing mutant tpk1 and also tpk2 and tpk3 deletions did not significantly alter its sensitivity to this DNA-damaging agent. Yeast transformants containing increased kinase activity resulting from overexpression of RAS2Val19 or TPK1 and yeast strains having increased kinase activities due to mutations in the BCY1 gene also did not show alterations in their sensitivity to cisplatin. Therefore, results from these studies unambiguously demonstrate that changes in PKA activity have no effect on cisplatin sensitivity in Saccharomyces cerevisiae.

Regulation of the multidrug resistance (MDR1) gene expression.

The emergence of drug resistance poses a major obstacle to the success of chemotherapy for a large number of human cancers. Development of the multidrug resistance phenotype in human malignancies is an especially pressing problem because the tumors become cross-resistant to multiple chemotherapeutic agents that are both chemically and physically unrelated. The increased resistance to multiple cytotoxic natural product chemotherapeutic drugs is due to overexpression of the mdr gene, which encodes a plasma membrane ATP-dependent efflux pump. Expression of P-glycoprotein is tissue specific and found in a number of normal tissues, including colon, small intestine, kidney, liver and adrenal gland, as well as in the capillaries of brain and testis. The precise physiological functions in these tissue localizations is unclear at present. Intense efforts in many laboratories currently are invested on elucidating the functions of P-glycoprotein and investigating mechanisms that regulate the mdr gene expression.

Characterization of a cAMP-dependent protein kinase mutant resistant to cisplatin.

The signal transduction pathway of cAMP, mediated by the cAMP-dependent protein kinase (PKA), is involved in the regulation of metabolisms, cell growth and differentiation and gene expression. Isolated PKA mutants from Chinese hamster ovary (CHO) cells were used in our laboratory to study the role of cAMP in the development of drug resistance in cancer. We have found that PKA mutants harboring a defective regulatory (RI alpha) subunit, but not the catalytic (C) subunit, are more resistant to the chemotherapeutic drug cisplatin. To clarify the role of PKA in cisplatin resistance, we have performed a step-wise selection with a CHO RI alpha subunit mutant cell line, 10248, for further resistance to cisplatin. A representative clone (10248/CDDP(R)-5) was used for further characterization. These cisplatin-resistant PKA mutant cells remained refractory to cAMP-induced growth inhibition and had decreased PKA activity comparable to the parental 10248 mutant cells. Furthermore, 10248/CDDP(R)-5 also exhibited cross-resistance to the nitrogen mustard melphalan but maintained the same sensitivity as wild-type cells to non-DNA-damaging agents such as methotrexate. The mechanism of resistance may be due to increased DNA repair as assessed by the host cell reactivation assay. We speculate that mutation and functional inactivation of PKA may result in deregulated growth response to cAMP, as well as the acquisition of resistance to cisplatin and other DNA-damaging agents in cancer.

Effects of RIalpha overexpression on cisplatin sensitivity in human ovarian carcinoma cells.

Our laboratory has found that Chinese hamster ovary (CHO) and mouse Y1 adrenocortical carcinoma PKA mutants with a defective R subunit, but not altered C subunits, exhibit increased resistance to cisplatin as well as other DNA-damaging agents. The mechanism of resistance may be associated with increased recognition of the cisplatin-damaged DNA and protein binding to the DNA lesion, thus enhancing DNA repair in the RI alpha mutants. These data suggest that mutation of RI alpha may confer resistance to cisplatin by affecting DNA repair activity. In the present study, we overexpressed RI alpha in human ovarian carcinoma A2780 cells to demonstrate that RI alpha can modulate cellular sensitivity to cisplatin. Retroviral-infected A2780 cells overexpressing wild-type RI alpha cDNA displayed a four- to eightfold greater sensitivity to cisplatin compared with parental cells. Overexpression of RI alpha in the CP70 cisplatin-resistant derivative of A2780 also increased the sensitivity of these cells to cisplatin. Therefore, enhanced expression of the RI alpha subunit of PKA sensitizes cells to the cytotoxic effects of this DNA-damaging agent. These data suggest that RI alpha may act directly, independent of the C subunit, to influence cellular sensitivity to cisplatin. Therefore, modulation of RI alpha expression or its functional status by pharmacological agents may potentially reverse cisplatin resistance in tumors.

Regulation of P-glycoprotein expression in cyclic AMP-dependent protein kinase mutants.

Multidrug resistance (MDR) in cancer poses a major obstacle to the success of chemotherapy. We previously reported that cyclic AMP (cAMP)-resistant mutants of the Chinese hamster ovary and the mouse adrenal cortical carcinoma cells harboring defective regulatory (RI alpha) subunits of the cAMP-dependent protein kinase (PKA) are more sensitive than wild-type cells to chemotherapeutic agents that are substrates for P-glycoprotein. In addition, a transfectant overexpressing a mutant RI alpha cDNA showed similar increased sensitivity to these drugs. The altered drug sensitivity in the RI alpha mutants results from reduced expression of the mdr gene, suggesting that PKA may regulate its expression. In this study, we evaluated the sensitivity of several Chinese hamster ovary catalytic (C) subunit mutants to various anticancer drugs. Like the RI alpha subunit mutant, the C subunit mutants also exhibit decreased kinase activity and unresponsiveness to growth inhibition by cAMP. However, in contrast to the RI alpha subunit mutant, the C subunit mutants are not multidrug sensitive and maintain P-glycoprotein expression levels comparable to those of wild-type cells. Furthermore, the C subunit mutants display the same resistance patterns as wild-type cells to P-glycoprotein substrates, including Adriamycin, Taxol, and colchicine. No significant difference was observed in their sensitivity to non-MDR drugs, such as 5-fluorodeoxyuridine, between wild-type, RI alpha, and C subunit mutant cells. These results suggest that the increased multidrug sensitivity in the PKA mutant cells results from alteration of the RI alpha subunit and not the kinase activity, thus implying novel functions for the RI alpha subunit. Therefore, genetic alteration of the RI alpha subunit of PKA may modulate drug resistance in cancer.

Involvement of phospholipase C in heat-shock-induced phosphorylation of P-glycoprotein in multidrug resistant human breast cancer cells.

The phosphorylation of P-glycoprotein has been appreciated for many years, yet little is known about the factors that initiate this post-translational modification. To determine whether the activation of P-glycoprotein phosphorylation could occur in response to cellular stress and to investigate the possible signal pathways involved, we studied the effect of heat shock on the phosphorylation of P-glycoprotein in sensitive and resistant MCF-7 human breast cancer cells. Treatment of multidrug resistant MCF-7/AdrR cells with heat shock increased the phosphorylation of P-glycoprotein. The response was not seen in the sensitive MCF-7 line, which does not express this drug transporter. Phosphorylation of P-glycoprotein induced by heat shock was not dependent on synthesis of new proteins, since phosphorylation was not inhibited by cycloheximide and the content of P-glycoprotein, as measured by immunoblotting, did not change after heat shock. The activation of P-glycoprotein phosphorylation by heat shock may be initiated through activation of phospholipase C, since heat shock stimulated the activity of this enzyme, as evidenced by increased formation of inositol trisphosphate and diacylglycerol and by phosphorylation of phospholipase C-gamma. U-73122, an inhibitor of phospholipase C and staurosporine, an inhibitor of protein kinase C, both decreased the heat-shock-induced phosphorylation of P-glycoprotein. These results suggest that heat shock induces phosphorylation of P-glycoprotein through the activation of the phospholipase C/protein kinase C pathway.

Stimulation of protein synthesis in pituitary cells by phorbol esters and cyclic AMP. Evidence for rapid induction of a component of translational initiation.

Phorbol esters such as phorbol myristate acetate (PMA) were employed to examine the involvement of protein kinase C in the regulation of protein synthesis in intact GH3 pituitary tumor cells. Amino acid incorporation increased as a function of time of pretreatment with these agents; 4-8- and 2-3-fold stimulations were observed for Ca2+-depleted and -restored preparations, respectively, following 2 h of exposure. PMA enhanced incorporation of amino acid into all detectable polypeptide species. Lysates of PMA-treated cells incorporated amino acid more efficiently than did lysates of untreated controls. Cells slowed at initiation by Ca2+ depletion responded to treatment with PMA with the production of low molecular weight polysomes and a concomitant decrease in 80 S monomers. In Ca2+-restored preparations, which form large polysomes, PMA treatment resulted in a decrease in 80 S monomers and a shift in average polysomal size from smaller to larger molecular weight. Ribosomal transit times, however, were not altered. PMA-stimulated amino acid incorporation and polysome formation were either eliminated or reduced significantly by actinomycin D and could not be ascribed to increased amino acid uptake or methionylation of tRNA. Substances which elevate cAMP in GH3 cells mimicked phorbol ester in its actions on protein synthesis. It is proposed that GH3 cells, in response to various stimuli, rapidly synthesize an mRNA that subsequently increases the synthesis of a rate-limiting component of translational initiation. Evidence that this pathway for translational control may function in alternative cell types is also presented.