The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance.

Induced expression of the Abcb1 drug transporter often occurs in tumors in response to chemotherapy. The role that epigenetic modifications within the ABCB1 promoter play in Abcb1 expression remains unclear. We selected MCF-7 cells for survival in increasing doses of chemotherapy drugs, and assessed the methylation status of 66 CpG sites within the ABCB1 promoter preceding, accompanying and following the onset of drug resistance. Increased ABCB1 transcript expression coincident with acquisition of resistance to epirubicin or paclitaxel was temporally associated with hypomethylation of the ABCB1 downstream promoter in the absence of gene amplifications or changes in mRNA stability. Treatment of control MCF-7 cells with demethylating and/or acetylating agents increased ABCB1 transcript expression. In addition to broad promoter hypomethylation, dramatic reductions in the methylation of specific CpG sites within the promoter were observed, suggesting that these sites may play a predominant role in transcriptional activation through promoter hypomethylation. Furthermore, our data suggest that allele-specific reductions in ABCB1 promoter methylation regulate promoter usage within paclitaxel-resistant cells. This study provides strong evidence that changes in ABCB1 promoter methylation, ABCB1 promoter usage and ABCB1 transcript expression can be temporally and causally correlated with the acquisition of drug resistance in breast tumor cells.

Evaluation of sICAM-1, sVCAM-1, and sE-Selectin levels in patients with metastatic breast cancer receiving high-dose chemotherapy.

Soluble forms of some cell adhesion molecules (CAM), sICAM-1, sVCAM-1, and sE-selectin, are elevated in the sera and plasma of patients with inflammation, arthritis, diabetes, and cancer. Increased levels of these soluble molecules in patients with cancer have been shown to correlate with disease progression and survival. This suggests that increased expression of the soluble forms of CAMs may play an important role in cancer cell growth and metastasis and may be prognostic and/or predictive of malignant disease. In this retrospective study, we assessed the clinical significance of sICAM-1, sVCAM-1, and sE-selectin in 95 patients with metastatic breast cancer enrolled in clinical trials of high-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT). The significance of soluble HER-2 (sHER-2) and sFAS status, determined in previous studies for this group of patients, was also included in this analysis. Univariate analysis showed that sICAM-1, sVCAM-1, sFas, sHER-2 positive status, and the presence of liver metastases were significant prognostic factors for both progression-free survival (PFS) and overall survival (OS) in the total patient group. In multivariable analysis, HER-2 and sFAS were shown to be independent prognostic factors for PFS and OS. Within the various treatment groups examined, sICAM-1 was a prognostic factor for clinical outcome for patients with metastatic breast cancer enrolled in trials with cyclophosphamide- and carboplatin-based or vinblastine-based HDC, but not in trials with paclitaxeland cyclophosphamide-based HDC.

HER-2 expression is a prognostic factor in patients with metastatic breast cancer treated with a combination of high-dose cyclophosphamide, mitoxantrone, paclitaxel and autologous blood stem cell support.

The expression levels of a circulating extracellular domain of HER-2 can be detected in the plasma and serum of patients with metastatic breast cancer using an enzyme immunoassay (ELISA) method. In this study, we evaluated the clinical significance of high and low levels of HER-2 in the plasma of 46 patients with metastatic breast cancer enrolled in a clinical trial of high-dose chemotherapy (HDCT) using cyclophosphamide, mitoxantrone, and paclitaxel with autologous stem cell transplantation (ASCT). Using 2500 U/ml as the cut-point, 20 patients (46%) had elevated HER-2 levels (HER-2 positive). Our results suggest that patients with metastatic breast cancer and high soluble plasma HER-2 have a significantly poorer overall (OS) and progression-free survival (PFS) following high-dose chemotherapy with paclitaxel and ASCT. The median OS of patients with low levels of HER-2 was significantly longer (P < 0.01) than the median OS of patients with high levels of HER-2 (29.8 months vs 15.9 months). PFS was also significantly longer (P < 0.01) for patients who were HER-2-negative, than for patients who were HER-2-positive (13.0 vs 8.6 months). Univariate analysis showed that patients with liver or lung metastases had significantly reduced OS and PFS. Patients with metastases to two or more sites also had a significantly reduced time to disease progression, but not OS. In multivariable analysis, lung metastases contributed along with HER-2-positive status to determine a group of patients with significantly poorer OS. However, HER-2-positive status remained the only independent predictor of PFS.

Soluble Fas (CD95) is a prognostic factor in patients with metastatic breast cancer undergoing high-dose chemotherapy and autologous stem cell transplantation.

The Fas/Fas ligand (FasL) system plays an important role in cellular apoptosis and is involved in cancer cell death induced by the immune system and anticancer drugs. Increased serum levels of soluble Fas (sFas) are associated with a number of different disease states and with tumor progression and metastasis in patients. In this study, we examined the plasma levels of sFas in 94 women with metastatic breast cancer undergoing high-dose chemotherapy (HDCT) treatment with autologous stem cell transplantation (ASCT) using a quantitative enzyme-linked immunosorbent assay (ELISA) method. Thirty-one patients (31/94, 33%) had plasma sFas levels greater than the optimum cut point of 1.90 ng/ml (median 2.47, range 1.98-13.54 ng/ml) and were designated as sFas positive. Sixty-three patients (63/94, 67%) had sFas levels below 1.90 ng/ml (median 1.14, range 0.47-1.89 ng/ml). In univariate analysis, patients with sFas-positive status, HER-2 overexpression, and the presence of liver metastases had a significantly shorter time to disease progression (PFS) and significantly decreased overall survival (OS). Multivariable analysis (Cox proportional hazards model) for PFS determined that sFas status significantly predicted disease progression (p = 0.004) with an adjusted hazard ratio (HR) of 2.0 (95% CI, 1.3-3.3). HER-2 status and liver metastases were also significant independent predictors of disease progression (p < 0.001) for both. sFas level was also an independent prognostic factor for OS with an adjusted HR of 2.0 (p = 0.006; 95% CI, 1.2-3.4). HER-2 status and liver metastases also remained highly significant independent prognostic factors for OS (HER-2: p < 0.001, HR 2.3, and liver metastases: p = 0.001, HR 2.7). In conclusion, these results suggest that plasma levels of sFas may be a valuable clinical prognostic factor in predicting outcome (PFS and OS) for patients with metastatic breast cancer undergoing HDCT with ASCT.

Role of specific apoptotic pathways in the restoration of paclitaxel-induced apoptosis by valspodar in doxorubicin-resistant MCF-7 breast cancer cells.

Paclitaxel (Taxol) kills tumor cells by inducing both cellular necrosis and apoptosis. A major impediment to paclitaxel cytotoxicity is the establishment of multidrug resistance whereby exposure to one chemotherapeutic agent results in cross-resistance to a wide variety of other drugs. For example, selection of MCF-7 breast cancer cells for resistance to doxorubicin (MCF-7ADR cells) results in cross-resistance to paclitaxel. This appears to involve the overexpression of the drug transporter P-glycoprotein which can efflux both drugs from tumor cells. However, MCF-7ADR cells possess a deletion mutation in p53 and have considerably reduced levels of the Fas receptor, Fas ligand, caspase-2, caspase-6, and caspase-8, suggesting that paclitaxel resistance may also stem from a bona fide block in paclitaxel-induced apoptosis in these cells. To address this issue, we examined the ability of the P-glycoprotein inhibitor valspodar to restore paclitaxel accumulation, paclitaxel cytotoxicity, and paclitaxel-induced apoptosis. Compared to drug sensitive MCF-7 cells, MCF-7ADR cells accumulated >6-fold less paclitaxel, were approximately 100-fold more resistant to killing by the drug, and were highly resistant to paclitaxel-induced apoptosis. In contrast, MCF-7ADR cells pretreated with valspodar were indistinguishable from drug-sensitive cells in their ability to accumulate paclitaxel, in their chemosensitivity to the drug, and in their ability to undergo paclitaxel-induced apoptosis. Valspodar, by itself, did not affect these parameters. This suggests that the enhancement of paclitaxel toxicity in MCF-7ADR cells involves a restoration of apoptosis and not solely through enhanced drug-induced necrosis. Morever, it appears that changes in the levels/activity of p53, the Fas receptor, Fas ligand, caspase-2, caspase-6, or caspase-8 activity have little effect on paclitaxel-induced cytotoxicity and apoptosis in human breast cancer cells.

Lack of modulation of MDR1 gene expression by dominant inhibition of cAMP-dependent protein kinase in doxorubicin-resistant MCF-7 breast cancer cells.

The drug transporter P-glycoprotein (P-gp) appears to play an important role in the ability of tumor cells to evade killing by chemotherapeutic agents. Using pharmacological inhibitors of cAMP-dependent protein kinase (PKA), it has been suggested that, similar to rodent model systems, the human P-gp gene (MDR1) is also under PKA-dependent control and that PKA inhibition may prove useful in reducing drug resistance in human cancer cells. To test this hypothesis, we stably transformed doxorubicin (Adriamycin)-resistant human MCF-7 breast cancer cells (MCF-7(ADR)) with a vector that inhibits PKA activity by inducing over-expression of mutant type Ialpha PKA regulatory (RIalpha) subunits. Two transformants (MCF-7(ADR-A) and MCF-7(ADR-B)) were found to express mutant RIalpha subunits and to possess markedly reduced PKA activity; another transformant (MCF-7(ADR-9)) lacked mutant RIalpha subunit expression and exhibited no inhibition of PKA activity. In contrast with findings in Chinese hamster ovary and Y1 adrenal cells, P-gp levels and cellular sensitivity to drugs which are P-gp substrates were unchanged in the PKA-inhibited transformants, suggesting that P-gp expression and function are not under PKA-dependent control in MCF-7(ADR) cells. Growth and saturation densities of the cell lines were highly correlated with level of PKA catalytic activity, suggesting that PKA inhibition may prove useful in inhibiting growth of breast tumor cells, even upon establishment of resistance to doxorubicin. However, our results challenge current proposals that drug sensitivity in P-gp-expressing human tumor cells may be restored by blocking MDR1 gene expression through inhibition of PKA activity.

Inhibitory properties of the regulatory domains of human protein kinase Calpha and mouse protein kinase Cepsilon.

Two fusion proteins in which the regulatory domains of human protein kinase Calpha (Ralpha; amino acids 1-270) or mouse protein kinase Cepsilon (Repsilon; amino acids 1-385) were linked in frame with glutathione S-transferase (GST) were examined for their abilities to inhibit the catalytic activities of protein kinase Calpha (PKCalpha) and other protein kinases in vitro. Both GST-Ralpha and GST-Repsilon but not GST itself potently inhibited the activities of lipid-activated rat brain PKCalpha. In contrast, the fusion proteins had little or no inhibitory effect on the activities of the Ser/Thr protein kinases cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, myosin light chain kinase, and mitogen activated protein kinase or on the src Tyr kinase. GST-Ralpha and GST-Repsilon, on a molar basis, were 100-200-fold more potent inhibitors of PKCalpha activity than was the pseudosubstrate peptide PKC19-36. In addition, a GST-Ralpha fusion protein in which the first 32 amino acids of Ralpha were deleted (including the pseudosubstrate sequence from amino acids 19-31) was an effective competitive inhibitor of PKCalpha activity. The three GST-R fusion proteins also inhibited protamine-activated PKCalpha and proteolytically activated PKCalpha (PKM), two lipid-independent forms of PKCalpha; however, the IC50 values for inhibition were 1 order of magnitude greater than the IC50 values obtained in the presence of lipid. These results suggest that part of the inhibitory effect of the GST-R fusion proteins on lipid-activated PKCalpha may have resulted from sequestration of lipid activators. Nonetheless, as evidenced by their abilities to inhibit the lipid-independent forms of the enzyme, the GST-R fusion proteins also inhibited PKCalpha catalytic activity through direct interactions. These data indicate that the R domains of PKCalpha and PKCepsilon are specific inhibitors of protein kinase Calpha activity and suggest that regions of the R domain outside the pseudosubstrate sequence contribute to autoinhibition of the enzyme.

Molecular strategies for the dominant inhibition of protein kinase C.

The regulatory (R) domain of PKC alpha fused to glutathione-S-transferase (GST-R alpha) competitively inhibited PKC activity associated with extracts of Y1 mouse adrenocortical tumor cells and the activities of several specific PKC isozymes. GST-R alpha did not inhibit the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase or calmodulin-dependent myosin light chain kinase. GST-R alpha inhibited PKC activities 20 times more potently than did a synthetic peptide corresponding to the pseudosubstrate sequence of PKC alpha. In intact yeast cells, the R domain prevented PKC beta-1-induced inhibition of growth and cytokinesis. These results indicate that the R domain of PKC alpha acts as a specific, dominant inhibitor of PKC activity, and suggest that the PKC alpha R domain may provide a useful genetic tool to assess the roles of PKC in various signal transduction processes.

Regulatory domain of human protein kinase C alpha dominantly inhibits protein kinase C beta-I-regulated growth and morphology in Saccharomyces cerevisiae.

This study demonstrates that the isolated regulatory (R) domain (amino acids 1-270) of human protein kinase C alpha (PKC alpha) is a potent inhibitor of PKC beta-I activity in a yeast expression system. The PKC alpha R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKC beta-I in vitro (Ki = 0.2 microns) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19-36 from PKC alpha. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKC alpha R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKC beta-I activity in vivo, in a yeast strain expressing rat PKC beta-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKC beta-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKC beta-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKC alpha acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes.

Identification of promoter elements in the mouse 21-hydroxylase (Cyp21) gene that require a functional cyclic adenosine 3',5'-monophosphate-dependent protein kinase.

The constitutive and cAMP-induced expression of the mouse steroid 21-hydroxylase gene (Cyp21) are impaired in adrenal cell mutants harboring mutations in cAMP-dependent protein kinase (cAMPdPK). These requirements for a functional cAMPdPK have been mapped to the proximal 330 basepairs of the Cyp21 promoter. This study attempts to identify specific promoter elements of Cyp21 that require cAMPdPK for constitutive activity by comparing their abilities to enhance the expression of a reporter gene in Y1 adrenocortical tumor cells and Y1 Kin mutants defective in cAMPdPK activity. As determined in transient transfection assays, Cyp21 promoter elements at -65, -140, -170, -210, and -280 each enhanced the expression of a human GH reporter gene in parent Y1 cells. The relative order of effectiveness of each of these elements was: -170 >> -280 > -140 > -65 > or = -210. The -170 element was 25-fold more effective in enhancing gene expression from the reporter construct in Y1 cells than in Kin mutant cells; the elements at -65, -140, and -210 were 3-fold more effective in Y1 cells than in Kin mutant cells; the -280 element was equally effective in the parent and Kin mutant clones. These studies suggest that the promoter elements at -170, -65, -140, and -210 mediate the requirement for a functional cAMPdPK in the expression of Cyp21. As determined by gel mobility shift assays with these elements, the dependence of the Cyp21 promoter elements on a functional cAMP-dependent protein kinase did not result from decreased expression or binding affinities of their respective DNA-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)