Biomedical subjects
M Dabholkar
Publications and source records attributed to M Dabholkar.
Paclitaxel, cisplatin, and cyclophosphamide in human ovarian cancer: molecular rationale and early clinical results.
The three most active types of agents in the treatment of cancer of the ovary are platinum compounds (cisplatin or carboplatin), bifunctional alkylating agents (cyclophosphamide, melphalan, etc), and the recently developed natural product paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ). In an effort to improve long-term disease-free survival in patients with advanced disease, we have developed a three-drug regimen consisting of cisplatin, paclitaxel, and cyclophosphamide. Granulocyte-colony stimulating factor is given as bone marrow support using a flexible dosing approach. The molecular basis for this approach is founded in the positive molecular interactions between cisplatin and bifunctional alkylating agents and between paclitaxel and DNA-damaging agents. Cisplatin and cyclophosphamide damage DNA by two very different mechanisms; the respective importance of DNA strand "kinking" versus DNA strand "cross-linking" may explain the positive cell kill interaction between these two drugs. Paclitaxel appears to markedly slow the repair of DNA lesions caused by DNA-damaging agents, which may include radiation-induced lesions, cisplatin adducts, and cyclophosphamide cross-links. Clinical data to date from several different groups strongly suggest that these molecular interactions translate into positive clinical benefit in human ovarian cancer. Preliminary data from our clinical trial show that these three agents are well tolerated in the doses administered and that this combination shows exceptional promise as a possible therapeutic advance in this disease.
Effects of interleukin-1 alpha on DNA repair in human ovarian carcinoma (NIH:OVCAR-3) cells: implications in the mechanism of sensitization of cis-diamminedichloroplatinum(II).
The cytokine interleukin-1 alpha (IL-1 alpha) showed a cytostatic effect on human ovarian carcinoma cells and significantly enhanced the antiproliferative activity of cis-diamminedichloroplatinum(II) (cisplatin) toward the NIH:OVCAR-3 tumor cell line in culture. The factor of sensitization was 15-20-fold. The maximum levels of sensitization were observed both with simultaneous exposure to cisplatin and IL-1 alpha and with 24-hr pretreatment with IL-1 alpha. Synergy between these agents was diminished when cells were pretreated with an IL-1 alpha-specific receptor antagonist, indicating that synergistic interaction was receptor mediated. Using atomic absorption spectroscopy, we evaluated the cellular accumulation of cisplatin and the DNA platination; the results showed that IL-1 alpha increased cellular accumulation of cisplatin and DNA platination. Cisplatin did not affect IL-1 alpha accumulation in NIH:OVCAR-3 cells. Further studies showed that IL-1 alpha reduced the removal of platinum from DNA. These results strongly suggest that IL-1 alpha inhibits DNA repair, and this decrease in DNA repair may explain, in part, the strong synergistic interaction between IL-1 alpha and cisplatin in NIH:OVCAR-3 cells.
Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy.
Nucleotide excision repair is a DNA repair pathway that is highly conserved in nature, with analogous repair systems described in Escherichia coli, yeast, and mammalian cells. The rate-limiting step, DNA damage recognition and excision, is effected by the protein products of the genes ERCC1 and XPAC. We therefore assessed mRNA levels of ERCC1 and XPAC in malignant ovarian cancer tissues from 28 patients that were harvested before the administration of platinum-based chemotherapy. Cancer tissues from patients whose tumors were clinically resistant to therapy (n = 13) showed greater levels of total ERCC1 mRNA (P = 0.059), full length transcript of ERCC1 mRNA (P = 0.026), and XPAC mRNA (P = 0.011), as compared with tumor tissues from those individuals clinically sensitive to therapy (n = 15). In 19 of these tissues, the percentage of alternative splicing of ERCC1 mRNA was assessed. ERCC1 splicing was highly variable, with no difference observed between responders and nonresponders. The alternatively spliced species constituted 2-58% of the total ERCC1 mRNA in responders (median = 18%) and 4-71% in nonresponders (median = 13%). These data suggest greater activity of the DNA excision repair genes ERCC1 and XPAC in ovarian cancer tissues of patients clinically resistant to platinum compounds. These data also indicate highly variable splicing of ERCC1 mRNA in ovarian cancer tissues in vivo, whether or not such tissues are sensitive to platinum-based therapy.
Platinum-DNA adduct in leukocyte DNA of a cohort of 49 patients with 24 different types of malignancies.
Using atomic absorbance spectrometry with Zeeman background correction, we measured platinum-DNA adduct levels in leukocyte DNA of 49 patients receiving therapy consisting of only carboplatin and cisplatin. Twenty-four histological types of malignancy were included in the cohort. Peripheral blood leukocytes were collected at defined times during the first two cycles of treatment. The relationship between adduct level and disease response was highly statistically significant during cycle 1 of therapy (two-sided P = 0.007 at day 2), but statistical significance was lost during cycle 2. On all days studied, median and mean adduct levels were consistently higher in responders as compared to nonresponders (summary two-sided P = 0.0004). These data suggest that the processes which protect cellular DNA may be common to malignant and nonmalignant rapidly dividing tissues of the same individual, regardless of the type of tumor that individual may harbor.
Expression of excision repair genes in non-malignant bone marrow from cancer patients.
The patterns of expression of 3 human DNA-repair genes (ERCC1, ERCC2, ERCC6) were assessed in 52 bone-marrow specimens obtained from cancer patients prepared for autologous bone-marrow transplantation. Marrow was collected prior to the initiation of treatment in patients with sarcoma or testicular cancer; marrow was collected after initial cytoreductive therapy for patients with non-Hodgkin's lymphoma, Hodgkin's disease, and other tumors. Slot-blot analysis of marrow RNA showed a bimodal pattern of ERCC1, ERCC2 and ERCC6 gene expression with relative expression values ranging more than 200-fold. This pattern was seen in all patient groups and appeared to be independent of whether or not patients had received prior chemotherapy. In all patient groups, when expression was low for ERCC1, expression was also low for ERCC2 and ERCC6, suggesting that expression of these genes may be coordinated within an individual although they are located on two different chromosomes. Southern blot analyses of Pst I digests of DNA from 6 bone-marrow samples indicate no differences in ERCC1 gene copy number between high expressors and low expressors. There is absence of restriction fragment length polymorphism for ERCC1 suggesting that the different levels of expression in high and low expressors were not due to major deletions or rearrangements of the ERCC1 gene. We conclude that expression of these ERCC genes may vary widely between individuals, and that within an individual, their expression may be linked and coordinated by a common regulatory mechanism.
ERCC1 and ERCC2 expression in malignant tissues from ovarian cancer patients.
BACKGROUND: ERCC1 and ERCC2 are human DNA repair genes that are associated with in vitro resistance to selected DNA-damaging agents. PURPOSE: Fresh tumor tissues from 26 patients with ovarian cancer were analyzed for the RNA levels of expression of these genes to determine possible clinical relevance. METHODS: Tumor tissues were harvested from patients immediately before they entered a cisplatin- or carboplatin-based treatment protocol. Clinical response was assessed by standard criteria. Gene expression level was assessed by slot blot analysis, using beta-actin as a control. Relative expression levels were determined by comparing each tumor sample with a Chinese hamster ovary cell line that had a stable transfection of the human ERCC1 gene. RESULTS: Patients who were clinically resistant to platinum-based therapy had a 2.6-fold higher expression level of ERCC1 in their tumor tissue than did patients who responded to that therapy (P = .015). Results obtained by slot blot analysis were qualitatively confirmed by polymerase chain reaction analysis. Relative levels of expression of ERCC2 did not differ significantly between responders and nonresponders. CONCLUSION: We conclude that ERCC1 expression levels in human tumor tissue may have a role in clinical resistance to platinum compounds. These data appear to be consistent with the assertion that ERCC1 serves as an excision nuclease, whereas ERCC2 serves as a helicase.
Host cell reactivation of cisplatin-damaged plasmid DNA in human non-T leukocyte cell lines.
Host-cell reactivation of cisplatin-damaged pRSVcat was assessed in three B cell lines (SKW 6.4, WIL2-NS, RPMI 1788), the monocytic cell line THP-1, and promyelocytic HL-60 cells. IC50 values following a 3-day exposure of the five leukocyte cell lines to cisplatin ranged from 0.45 to 1.92 microM. Transfer of pRSVcat into all cell lines was effected by electroporation and the resultant CAT activity was measured 24 h later by a rapid single vial CAT assay. CAT activity corresponding to an average of 0.06 units of purified CAT enzyme was expressed by WIL2-NS cells. Very low to no expression of the CAT vector was observed in all other cell lines studied, despite the presence of intracellular levels of 3H-labelled pRSVcat comparable to WIL2-NS. Epstein-Barr virus transformed B cells (SKW 6.4 and RPMI 1788) did not successfully perform host cell reactivation. In WIL2-NS cells, platination of pRSVcat to defined levels of 5-40 platinum molecules per plasmid led to a graded reduction in CAT activity expressed following transfection. Platination levels of 20 and 40 platinum molecules per plasmid did not alter the efficiency of transfer of pRSVcat into these cells by electroporation. Data obtained in this study suggests that EBV transformation may possibly be a negative influence on host cell-reactivation assays for cisplatin-DNA damaged plasmid in non-T human leukocytes.
Determinants of cisplatin sensitivity in non-malignant non-drug-selected human T cell lines.
We have studied molecular mechanisms of cisplatin sensitivity and resistance in 3 non-malignant, non-drug-selected human T lymphocyte cell lines. HuT 78, H9, and MOLT-4 cells were assessed for sensitivity to cisplatin, DNA damage levels following defined drug exposures, drug accumulation, and DNA repair efficiency as measured by adduct removal from cellular DNA and by host-cell reactivation of cisplatin-modified plasmid DNA. Based on 3-day continuous drug exposures, the IC50 values for the cell lines were: HuT 78, 0.83 microM; H9, 0.45 microM; and MOLT-4, 0.33 microM. These cells retained this order with respect to DNA repair capability, whether measured by platinum-DNA adduct removal from cellular DNA or by host-cell reactivation assays. DNA repair values measured by these two assays were directly related to one another with a linear correlation coefficient of 0.993. At sublethal cisplatin doses the more resistant cells showed the highest levels of drug uptake. When drug uptake levels were 'corrected' for drug-induced cell kill, there were equal levels of DNA repair efficiency for a given level of drug uptake. Absolute levels of cisplatin-DNA adduct repair increased with increasing drug dose. However, at supralethal doses of drug, efficient DNA repair could be overcome in all 3 cell lines with percentage-adduct-removal dropping from a 60-80% range to a less than 30% range. We conclude that in non-malignant non-drug-selected human T cells, DNA repair appears to be the primary determinant of cisplatin sensitivity/resistance and that enhanced DNA repair may be a biologic compensatory mechanism for cells that cannot prevent cellular uptake of DNA-damaging agents.
Cisplatin-DNA damage and repair in peripheral blood leukocytes in vivo and in vitro.
We have extended our studies on the relationship between cisplatin/carboplatin-induced DNA damage in readily accessible tissue(s) and clinical response to therapy. Such an approach may assist in the study of cancer drug resistance and in establishing parameters for assessing human populations for sensitivity to DNA damaging agents in the environment. Platinum-DNA adduct levels were measured by atomic absorbance spectrometry. DNA repair capacity was assessed in human T-lymphocytes by the ability to repair cisplatin lesions in cellular DNA or in transfected plasmid DNA. In a "blinded" study of 21 patients receiving combination cisplatin/carboplatin drug therapy, there was a direct relationship between DNA damage in leukocytes and disease response (summary two-sided p = 0.00011). The cohort of patients had 15 different tumor types, suggesting that blood tissue and tumor tissue of an individual may process platinum-DNA damage similarly regardless of the tissue of origin of the tumor. In leukocytes in vivo, persistence and accumulation were prominent features of the cisplatin-DNA adduct profile. Functional DNA repair capacity has been studied in eight human leukocyte cell lines in vitro (three, T-cells; three, B-cells; one, monocytic; one, promyelocytic), using a host cell reactivation assay with cisplatin-damaged pRSVcat. In the three T cell lines studied, host cell reactivation efficiency was directly related to the cells' abilities to repair cisplatin-damaged cellular DNA (correlation coefficient = 0.993).(ABSTRACT TRUNCATED AT 250 WORDS)
Host-cell reactivation of cisplatin-damaged pRSVcat in a human lymphoid cell line.
A method has been developed for studying host-cell reactivation of cisplatin-damaged plasmid DNA in human T lymphocytes. Parameters of electroporation were established for transfection of the shuttle vector pRSV cat into H9 cells, and a rapid single-vial assay was used for measurement of chloramphenicol acetyltransferase (CAT) activity in extracts of transfected cells. pRSVcat was modified with cisplatin to defined levels ranging from 5 to 40 platinum molecules per plasmid, and then transfected into H9 cells. Graded reductions in CAT activity were observed with increasing levels of platination of the plasmid. At 40 platinum molecules per plasmid, CAT activity was reduced to levels of the negative controls. The efficiency of electroporation-mediated transfer of plasmid into H9 cells was not reduced by high levels of cisplatin modification. The level of modification effecting a 63% reduction in CAT gene expression (the B0), was found to be 17 cisplatin molecules per plasmid. This assay system offers a rapid and sensitive method for assessing the capability of human lymphoid cells to reactivate cisplatin-damaged plasmid DNA.
Studies on natural killer cells in chronic myeloid leukemia patients in remission.
In our earlier studies, the natural killer (NK) cell mediated cytotoxicity was found to be impaired in 55% of chronic myeloid leukemia (CML) patients in remission (low NK responders), while the rest exhibited normal range of cytotoxicity (normal NK responders). In the present investigations probable factors contributing to the impaired NK activity of low NK responder CML patients have been analyzed. Peripheral blood lymphocytes (PBL) from both low and normal NK responder CML patients possessed normal percentages of HNK-1+, CD3+ and CD8+ cells. The proportion of HNK-1+ cells and CD8+ cells in low density fractions (LDF) and high density fractions (HDF) respectively of Percoll separated PBL from low and normal NK responder CML patients and healthy donors was comparable. However, the NK activity of LDF cells of low NK responder CML patients was much lower. Also, HDF cells from low NK responder CML patients exhibited a regulatory effect on NK cytotoxicity of PBL from healthy donors. Therefore, it is likely that the presence of suppressor cells and perhaps an intrinsic inability of HNK-1+ cells may together contribute to the impaired NK cytotoxicity of low NK responder CML patients.
Modulation of natural killer and antibody-dependent cellular cytotoxicity by interferon and interleukin-2 in chronic myeloid leukemia patients in remission.
Our earlier studies demonstrated that about 55% of chronic myeloid leukemia (CML) patients in remission exhibited impaired natural killer (NK) cytotoxicity (low NK responders) while antibody-dependent cellular cytotoxicity (ADCC) of these patients, on chicken red blood cells as targets, was within normal range. In this paper, we have attempted to modulate the NK cytotoxicity of CML patients in remission with interferon (IFN) and interleukin-2 (IL-2) singly or together. ADCC using K562 target-directed monoclonal antibody (MAb) 4.6E10 was also modulated by treating the effectors with IFN or IL-2. Pretreatment of nonadherent mononuclear cells from peripheral blood (NAPBMNC) with IFN or IL-2 was found to result in 20 and 21% increase in target cell lysis in case of healthy donors, 79 and 98% increase in case of CML normal NK responders, and 164 and 159% increase in case of CML low NK responders. Combined use of IFN and IL-2 potentiated further the lymphocytotoxicity to 25% in healthy donors, 135% in normal NK responder CML patients and 283% in low NK responder CML patients. This treatment resulted in restoration of cytotoxicity of the latter group of patients to a normal level. The augmentation was seen in 80-100% CML patients. Although ADCC with chicken red blood cells as targets was within normal range, ADCC mediated with MAb to K562 cells was significantly lower in CML low NK responders (24.5%) than CML normal NK responders (42.4%) and healthy donors (65.9%).(ABSTRACT TRUNCATED AT 250 WORDS)
Natural and antibody-dependent cellular cytotoxicity in chronic myeloid leukemia patients in remission.
Non-adherent peripheral blood mononuclear cells (NA-PBMNC) from 67 chronic myeloid leukemia (CML) patients in the first and two subsequent remissions, and 23 normal healthy donors were tested for NK and ADCC activities in short term chromium release assays using K562 and antibody-coated chicken RBCs as respective targets. CML patients in remission exhibited significantly reduced NK cytotoxicity (16. 1-19.7%) compared to normal healthy donors (47.4%). Of the patients tested, 55% exhibited NK levels below the mean percent cytotoxicity--2SD (12.5%) of normal donors (low responders), while 45% exhibited NK cytotoxicity above the 12.5% level (normal responders). On the other hand, CML patients in remission showed ADCC activity comparable to that of normal healthy donors (53.3%) irrespective of whether they belonged to normal NK responder group (55.5-65.0% ADCC) or low NK responder group (39.4-48.3% ADCC). The low or normal NK responder status of CML patients was not found to be related to either progression on the disease, or the type of drug used to bring about remission, or to the period in remission at the time of testing. In-vitro treatment of effector lymphocytes with recombinant human IFN alpha resulted in augmented of NK activity in both low and normal NK responder patients. The IFN-augmented NK activity in low responder patients however remained below the normal levels.