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Biomedical subjects

D Fink

Publications and source records attributed to D Fink.

At least 55 records · Page 3Linked to original sources
PubMed1998

Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase.

Past studies have documented the promise of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) suicide gene therapy as a potential antitumor treatment. HSV-TK converts the pro-drug ganciclovir (GCV) into a toxic nucleotide analogue, the incorporation of which into cellular DNA blocks cell proliferation. In this report, we have examined the hypothesis that the effectiveness of HSV-TK suicide gene therapy can be enhanced by coexpression of the antitumor cytokine human tumor necrosis factor-alpha (TNF-alpha) from the same replication-defective HSV-1 vector. In vitro testing demonstrated that TNF-alpha expression from this vector potentiated the killing of both TNF-alpha-sensitive L929 tumor cells and TNF-alpha-resistant U-87 MG cells in the presence of GCV. Furthermore, treatment of established intradermal L929 tumors in vivo with the TNF-alpha/TK vector and GCV resulted in prolonged animal survival compared with treatment with parental HSV-TK vector in the presence or absence of GCV. Treatment of intracerebral U-87 MG tumors showed a clear benefit of TK therapy, but a significant further increase in survival using the TNF-alpha vector could not be demonstrated. We found that potentiation of cell killing in vitro required intracellular TNF-alpha because purified protein added to the culture medium of cells infected with HSV-TK vector failed to have the same effect. Accordingly, potentiation in vivo should depend on efficient infection, but immunohistochemical analysis indicated that virus administration by U-87 MG intratumoral injection was inadequate, resulting in an estimated <1% infection of all tumor cells. Moreover, the majority of infected tumor cells were localized at the tumor margin. Together, these results suggest that TNF-enhanced tk gene therapy should provide a useful treatment for TNF-alpha-sensitive tumors and perhaps also for TNT-alpha-resistant tumors if vector delivery can be improved to increase the percentage of transduced tumor cells.

Animals↗
PubMed1998

Enrichment for DNA mismatch repair-deficient cells during treatment with cisplatin.

In addition to playing a role in tumorigenesis, loss of DNA mismatch repair results in low-level intrinsic resistance to cisplatin and carboplatin. We used a mismatch repair-deficient (clone B) and -proficient (clone B/rev) pair of Chinese hamster ovary sublines to determine the ability of cisplatin to enrich for repair-deficient cells during growth in vitro and in vivo. Clone B cells were 1.8-fold resistant to cisplatin as measured by a clonogenic assay. These cells were molecularly engineered to express constitutively the green fluorescent protein, and changes in the fraction of these repair-deficient cells were monitored by flow cytometric analysis. A single 1-hr exposure to cisplatin at an IC50 concentration enriched populations initially containing either 5 or 10% clone B cells by 81 and 75%, respectively, when measured at 5 days. Enrichment increased as a function of drug concentration to 158 and 169%, respectively, following an IC90 exposure. When grown as a xenograft, a single LD10 dose of cisplatin enriched the tumors by 48% from 4.6 to 6.8% repair-deficient cells (p = 0.04). To determine whether similar enrichment occurs during the treatment of human ovarian cancer patients, paired tumor samples were obtained from 38 patients before and after treatment with a minimum of 3 cycles of platinum drug-based primary chemotherapy and analyzed immunohistochemically for changes in the fraction of tumor cells expressing hMHL1. Following treatment there was a reduction in hMLH1 staining in 66% of the cases (p = 0.0005). Our results demonstrate that, despite the fact that loss of mismatch repair yields only modest levels of cisplatin resistance, even a single exposure to cisplatin produces quite a marked enrichment for repair-deficient cells in vitro and in vivo. Our results are consistent with the concept that treatment with cisplatin or carboplatin selects for preexisting mismatch repair-deficient cells, and that this contributes to the frequent development of clinical resistance.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The effect of different chemotherapeutic agents on the enrichment of DNA mismatch repair-deficient tumour cells.

Loss of DNA mismatch repair is a common finding in hereditary non-polyposis colon cancer as well as in many types of sporadic human tumours. We compared the effect of loss of DNA mismatch repair on drug sensitivity as measured by a clonogenic assay with its effect on the ability of the same drug to enrich for mismatch repair-deficient cells in a proliferating tumour cell population. Mixed populations containing 50% DNA mismatch repair-deficient cells constitutively expressing green fluorescent protein and 50% mismatch repair-proficient cells were exposed to different chemotherapeutic agents. 6-Thioguanine, to which DNA mismatch repair-deficient cells are known to be resistant, was included as a control. The results in the cytotoxicity assays and in the enrichment experiments were concordant. Treatment with either carboplatin, cisplatin, doxorubicin, etoposide or 6-thioguanine resulted in enrichment for mismatch repair-deficient cells, and clonogenic assays demonstrated resistance to these agents, which varied from 1.3- to 4.8-fold. Treatment with melphalan, paclitaxel, perfosfamide or tamoxifen failed to enrich for mismatch repair-deficient cells, and no change in sensitivity to these agents was detected in the clonogenic assays. These results identify the topoisomerase II inhibitors etoposide and doxorubicin as additional agents for which loss of DNA mismatch repair causes drug resistance. The concordance of the results from the two assay systems validates the enrichment assay as a rapid and reliable method for screening for the effect of loss of DNA mismatch repair on sensitivity to additional drugs.

Adenocarcinoma↗
PubMed1998

The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase.

The 38-amino-acid isoform of pituitary adenylate cyclase-activating polypeptide (PACAP38) elicits a robust outgrowth of neurites in cultured PC12 cells. Initiation of neurite outgrowth occurs within 4-8 hr after the addition of PACAP38. Treatment with PACAP38 does not elicit collateral activation of p140(trk) nerve growth factor receptor tyrosine kinase activity, nor is it associated with tyrosine phosphorylation of suc1-associated neurotrophic factor target, a selective target of neurotrophin tyrosine kinase receptors. Coadministration of epidermal growth factor with PACAP38 elicits an enhanced response. Induction of neurites is also observed on the addition of PACAP38 to dominant negative Src and Ras PC12 cell variants. PACAP38 stimulates extracellular signal-regulated kinase (Erk) activity >10-fold within 5 min, and the effect is augmented by cotreatment with epidermal growth factor. Pretreatment with the cAMP-dependent protein kinase-selective inhibitor, H-89, is ineffective as an antagonist of PACAP38-induced neurite outgrowth, whereas down-regulation of protein kinase C (PKC) by phorbol ester or incubation with PKC-selective inhibitors GF109203X and calphostin C effectively blocks PACAP38-stimulated neurite formation. Stimulation of Erk activity is inhibited by incubation with PD90859, a pharmacological antagonist of the threonine/tyrosine dual-specificity Erk. Inhibition of ligand-stimulated Erk activation prevents PACAP38-induced neurite outgrowth. Collectively, these findings indicate that PACAP38-stimulated neuritogenesis requires PKC and Erk activation but is independent of cAMP-dependent protein kinase, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase.

Animals↗
PubMed1998

The role of DNA mismatch repair in drug resistance.

Loss of DNA mismatch repair (MMR) has been observed in a variety of human cancers. In addition to predisposing to oncogenesis, loss of MMR activity is of concern with respect to the use of chemotherapeutic agents to treat established tumors. Loss of MMR results in drug resistance directly by impairing the ability of the cell to detect DNA damage and activate apoptosis and indirectly by increasing the mutation rate throughout the genome. The MMR proteins are involved in mediating the activation of cell cycle checkpoints and apoptosis in response to DNA damage. MMR-deficient cells have been reported to be resistant to the methylating agents procarbazine and temozolomide, the alkylating agent busulfan, the platinum-containing drugs cisplatin and carboplatin, the antimetabolite 6-thioguanine, and the topoisomerase II inhibitors etoposide and doxorubicin. In the case of cisplatin, busulfan, temozolomide, and procarbazine, the degree of resistance has been shown to be sufficient to produce a large difference in clinical responsiveness in vivo in tumor model systems. The available preclinical data suggest that tumors that contain a significant fraction of cells deficient in MMR will demonstrate reduced responsiveness to specific drugs. The challenge now is to assess the clinical significance of the presence of deficient cells in tumors and to discover drugs that retain activity against MMR-deficient cells.

Alkylating Agents↗
PubMed1997

Loss of DNA mismatch repair: effects on the rate of mutation to drug resistance.

BACKGROUND: The loss of the ability of cells to repair mismatches in double-stranded DNA is a common finding in human tumors. This defect results in genomic instability and in increased resistance to several of the drugs used in cancer chemotherapy. The human colon cancer cell line HCT116 is deficient in DNA mismatch repair (MMR) because of a genetic defect in the hMLH1 gene, which is located on chromosome 3. In this study, we investigated whether MMR-deficient HCT116+chr2 cells (i.e., HCT116 cells into which chromosome 2 has been transferred [as a control]) have a higher rate of mutation to resistance to commonly used chemotherapeutic agents (i.e., cisplatin, doxorubicin, paclitaxel [Taxol], and etoposide) than MMR-proficient HCT116+chr3 cells (i.e., HCT116 cells into which chromosome 3 has been transferred to provide a wild-type copy of the hMLH1 gene). METHODS: Spontaneous mutation rates were calculated from measurements of the mutant fractions of cells before and after their expansion through a known number of generations (also known as the technique of maximum likelihood estimation). Aliquots of 500000 cells were expanded in culture over a period of 2 weeks, and the mutant fractions were determined both before and after expansion of secondary cultures (each also with an initial 500000 cells) in drug concentrations that produced survival fractions of 0.0002%. RESULTS: Mutation rates in MMR-proficient and MMR-deficient cells did not differ on exposure to cisplatin, doxorubicin, or paclitaxel; however, the relative mutation rate was 2.4-fold higher in MMR-deficient cells exposed to etoposide (P=.002). CONCLUSION: These results suggest that genes involved in the control of cellular sensitivity to etoposide are targets for mutation when the loss of MMR destabilizes the genome. Tumors containing large fractions of MMR-deficient cells may demonstrate more rapid emergence of clinical resistance to etoposide.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin.

The c-Abl nonreceptor tyrosine kinase and the c-Jun NH2-terminal kinase (JNK/stress-activated protein kinase) are activated during the injury response to the DNA-damaging agent cisplatin. Loss of DNA mismatch repair activity results in resistance to cisplatin in human cancer cells, suggesting that the mismatch repair proteins function as a detector for cisplatin DNA adducts. To identify signaling pathways activated by this detector, we investigated the effect of the loss of DNA mismatch repair function on the ability of cisplatin to activate the JNK and c-Abl kinases. The results demonstrate that cisplatin activates JNK kinase 3.8 +/- 0.2-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that activation of c-Abl is completely absent in the DNA mismatch repair-deficient cells. Furthermore, the results show that cisplatin-induced activation of JNK occurs through a stress-activated protein kinase/extracellular signal-regulated kinase kinase 1-independent mechanism. We conclude that activation of JNK and c-Abl by cisplatin is in part dependent upon the integrity of DNA mismatch repair function, suggesting that these kinases are part of the signal transduction pathway activated when mismatch repair proteins recognize cisplatin adducts in DNA.

Adenocarcinoma↗
PubMed1997

In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair.

In vitro studies have shown that loss of DNA mismatch repair due to lack of either hMSH2 or hMLH1 activity results in low-level resistance to cisplatin but not to oxaliplatin, an analogue that produces a different type of DNA adduct. No information is currently available on whether this low-level resistance is sufficient to result in enrichment of mismatch repair-deficient cells during drug exposure in vitro or to account for clinical failure of treatment in vivo. Mixed populations of cells containing a minority of DNA mismatch repair-deficient cells constitutively expressing green fluorescence protein were exposed repeatedly in vitro to cisplatin and oxaliplatin. Treatment with cisplatin resulted in a gradual enrichment for DNA mismatch repair-deficient cells, whereas treatment with oxaliplatin did not. MSH2-/- and MSH2+/+ embryonic stem cells were established as xenografts in athymic nude mice. Animals were treated 48 h after tumor implantation with a single LD10 dose of either cisplatin or oxaliplatin. MSH2-/- tumors were significantly less responsive to cisplatin than MSH2+/+ tumors, whereas there was no difference in sensitivity to oxaliplatin. These results demonstrate that the degree of cisplatin resistance conferred by loss of DNA mismatch repair is sufficient to produce both enrichment of mismatch repair-deficient cells during treatment in vitro and a large difference in clinical responsiveness in vivo. The results identify loss of DNA mismatch repair as a mechanism of resistance to cisplatin but not oxaliplatin.

Adenocarcinoma↗
PubMed1997

Down-regulation of epidermal growth factor receptors by nerve growth factor in PC12 cells is p140(trk)-, Ras-, and Src-dependent.

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells.

Animals↗
PubMed1997

Endometrioid ovarian carcinoma in a premenarchal girl: report of a case.

In children and adolescents, ovarian neoplasms are predominantly germ cell and sex cord stromal tumors. Carcinomas are quite rare, and, in particular, endometrioid adenocarcinomas are extremely rare in this age group. We report the case of a 13-year-old girl with FIGO stage I, grade I endometrioid adenocarcinoma of the ovary. To our knowledge this is the first report of an endometrioid carcinoma of the ovary occuring in the premenarchal age group and only the second case reported before age 15. Our patient has been treated by conservative surgery without postoperative chemotherapy. Menarche occured 3 months after surgery. Twelve months after surgery she is free of disease.

Adolescent↗
PubMed1997

Expression of the DNA mismatch repair proteins hMLH1 and hPMS2 in normal human tissues.

hMLH1 and hPMS2 are part of the DNA mismatch repair complex. Mutations in these genes have been linked to hereditary non-polyposis colon cancer; they also occur in a variety of sporadic cancers. Western blot analysis and immunohistochemistry demonstrated that hMLH1 and hPMS2 are widely expressed nuclear proteins with a distribution pattern very similar to that previously described for hMSH2. These observations showing similar localization of hMLH1 and hPMS2 with hMSH2 are consistent with the biochemical function of these proteins in DNA mismatch repair.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Discordance in p53 mutations when comparing ascites and solid tumors from patients with serous ovarian cancer.

We performed a mutational analysis of the p53 gene in matched samples of solid tumors and ascites from patients with ovarian cancer using the single-strand conformation polymorphism technique on exons 5-9 of the p53 gene on fresh and cultured material. We observed a discordance in the pattern of p53 mutations between the ascites and their solid tumor counterpart. In two cases, cancer cells from ascites carried a p53 mutation and the corresponding solid tumor cells retained the wild-type allele, while the opposite pattern was observed in two other patients samples. These results suggest that ovarian tumor cells within the ascites may not simply represent cells shed from the ovarian solid tumor and bear directly on gene therapy strategies in ovarian cancer.

Aged↗
PubMed1997

Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells.

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed1997

all-trans retinoic acid enhances cisplatin-induced apoptosis in human ovarian adenocarcinoma and in squamous head and neck cancer cells.

Cisplatin exerts its cytotoxicity by inducing apoptosis. Similarly, all-trans retinoic acid (ATRA) causes apoptosis in certain cells. We studied the interaction of cisplatin and ATRA in human ovarian adenocarcinoma cells 2008, in human head and neck squamous carcinoma cells UMSCC10b, and in their respective cisplatin-resistant sub-lines. ATRA enhanced the cytotoxicity of cisplatin. The interaction of the drugs was synergistic in combination index-isobologram analyses (combination index >0.5 at 50% cell survival) in all of the cell lines tested. ATRA inhibited the cellular accumulation of the cisplatin analogue [3H] cis-dichloroethylenediamineplatinum(II) by 22-33% in three of four cell lines tested but did not alter the cellular content of reduced glutathione. The expression of Bcl-2 relative to Bax decreased more after combined treatment with cisplatin and ATRA than after either drug alone. The apoptotic mechanism of cell death was confirmed by demonstrating cleavage of poly(ADP-ribose)polymerase and by morphological analysis. The combined treatment with ATRA and cisplatin induced apoptosis in significantly more cells than either drug alone. We conclude that ATRA enhances the cytotoxicity of cisplatin by facilitating apoptosis in ovarian and head and neck carcinoma cells.

Adenocarcinoma↗
PubMed1997

Pharmacological basis for a novel therapeutic strategy based on the use of aquated cisplatin.

In pursuit of a strategy for increasing delivery of platinum drugs to tumors, we compared the cytotoxicity, extent of cellular uptake, and DNA platination of native cisplatin (DDP) and aquated cisplatin (aqDDP) in human head and neck carcinoma UMSCC10b cells. AqDDP was 1. 8-fold more toxic than DDP when tested against UMSCC10b cells in vitro. At high concentrations, aqDDP uptake was 3-fold more rapid than that of DDP; uptake of DDP and aqDDP was nonsaturable up to a concentration of 1600 micrometer. AqDDP produced 6.4-fold more platination of DNA than did DDP at the same concentration, suggesting that once inside the cell, aqDDP was 2-fold more effective at producing adducts in DNA than the native drug. Despite the paradox that aqDDP, which contains some charged species, entered the cell more rapidly than did neutral native DDP at high concentrations, studies on the effect of temperature, ATP depletion, and sulfhydryl group blockade did not provide evidence for uptake of aqDDP via a channel or transporter. AqDDP was more nephrotoxic to mice than DDP; however, s.c. administration of sodium thiosulfate protected against this toxicity and permitted a 7-fold escalation of aqDDP dose. These studies provide the preclinical basis for a novel therapeutic strategy based on the regional intraarterial or intracavitary administration of aqDDP in combination with a systemic neutralizing agent.

Adenosine Triphosphate↗
PubMed1997

In vitro antineoplastic activity of a novel lanthionine-containing peptide.

It has been a long-term goal to discover peptides that can kill tumor cells while sparing normal tissues. Lan-7 is a novel, chemically stable peptide structurally related to somatostatin that contains a lanthionine bridge between the two cysteines in the peptide; TT-232 is a less stable analogue containing a disulfide bridge. The antitumor activity of Lan-7 was examined, relative to that of TT-232 and the clinically used analogue octreotide, against a panel of malignant human tumor cell lines and normal human hematopoietic precursors. Lan-7 was cytotoxic to all four tumor cell lines, with IC50 values ranging over a 2-fold range from 16 to 36 microM. The potency of Lan-7 was comparable to that of TT-232, and both of these agents were two to three times more potent than octreotide. At concentrations that were highly cytotoxic to tumor cells, Lan-7 produced no significant toxicity to normal human hematopoietic precursors. Lan-7 induced apoptosis in human ovarian carcinoma 2008 cells over the same concentration range at which it produced cytotoxicity, but it did so without activating G1, S, or G2 checkpoints, given that it produced no perturbation of cell cycle phase distribution. Cells engineered to overexpress P-glycoprotein were not more resistant to Lan-7 than isogeneic cells not expressing the mdr1 gene. These results make Lan-7 of interest as a potential cancer chemotherapeutic agent.

ATP Binding Cassette Transporter, Subfamily B, Mem↗