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Biomedical subjects

A B Pardee

Publications and source records attributed to A B Pardee.

At least 37 records · Page 2Linked to original sources
PubMed2000

Suberoylanilide hydroxamic acid as a potential therapeutic agent for human breast cancer treatment.

BACKGROUND: Suberoylanilide hydroxamic acid (SAHA) is a prototype of the newly developed, second-generation, hybrid polar compounds. It is a novel histone deacetylase inhibitor with high potency for inducing cell differentiation of cultured murine erythroleukemia cells. Studies with SAHA have primarily been performed with hematopoietic tumor cells. Here we extent these studies with SAHA to human breast cancer cell lines in an attempt to find better therapeutic agents for breast cancer treatment. MATERIALS AND METHODS: Human breast cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-435, as well as normal cells, including the normal breast epithelial cell line MCF-10A, and fibroblasts, were treated with SAHA. Cells assayed for cell survival by using trypan blue exclusion assay, colony formation assay, and cell cycle and apoptosis analysis. The effects of SAHA on cell cycle and apoptosis regulatory proteins were examined by Western blots analysis. The identification of additional target genes was carried out by differential display (DD) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: SAHA inhibited clonogenic growth of MCF7, MDA-MB-231, and MDA-MB-435 breast cancer cells. These cells were more sensitive to SAHA-mediated cytotoxic effects than normal breast epithelial cells and fibroblasts. The cytotoxic effects of SAHA on breast cancer cells were manifested by G1 and G2/M cell cycle arrest and eventual apoptosis. The pan-caspase inhibitor, Z-VAD.fmk, blocked SAHA-induced cell death, DNA laddering, and cleavage of poly(ADP-ribose) polymerase, indicating the involvement of caspases in SAHA-mediated apoptosis. In addition, SAHA modulated cell cycle and apoptosis regulatory proteins. For example, cyclin-dependent kinase (CDK) inhibitors p21WAF1/Cip1 and p27Kip1 were induced, and retinoblastoma protein pRb was hypophosphorylated. Moreover, SAHA induced several genes associated with differentiation and/ or growth inhibition. These genes encode gelsolin, isopentenyl-diphosphate delta isomerase (IDI1), and 1,25-dihydroxyvitamin D-3 up-regulated protein 1 (VDUP1), the last two of which were identified by DD. Induction of these genes may contribute to SAHA-mediated pro-differentiating and antiproliferative effects. CONCLUSIONS: SAHA induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer cells, possibly by modulating cell cycle and apoptosis regulatory proteins, such as CDK inhibitors p21 and p27, pRb, and other differentiation and/or growth inhibition-associated genes, including gelsolin, IDI1 and VDUP1. This, together with the low toxicity in normal cells, suggests that SAHA might have therapeutic potential for the treatment of human breast cancers.

Antineoplastic Agents↗
PubMed2000

Potent induction of apoptosis by beta-lapachone in human multiple myeloma cell lines and patient cells.

BACKGROUND: Human multiple myeloma (MM) remains an incurable hematological malignancy. We have reported that beta-lapachone, a pure compound derived from a plant, can induce cell death in a variety of human carcinoma cells, including ovary, colon, lung, prostate, pancreas, and breast, suggesting a wide spectrum of anticancer activity. MATERIALS AND METHODS: We first studied antisurvival effects of beta-lapachone in human MM cells by colony formation assay. To determine whether the differential inhibition of colony formation occurs through antiproliferative activity, we performed MTT assays. The cytotoxicity of beta-lapachone on human peripheral blood mononuclear cells was also measured by MTT assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the propidium iodide staining procedure to determine the sub-GI fraction, Annexin-V staining for externalization of phosphatidylserine, and fragmentation of cellular genomic DNA subjected to gel electrophoresis. To investigate the mechanism of anti-MM activity, we examined Bcl-2 expression, cytochrome C release, and poly (ADP ribose) polymerase cleavage by Western blot assay. RESULTS: We found that beta-lapachone (less than 4 microM) inhibits cell survival and proliferation by triggering cell death with characteristics of apoptosis in ARH-77, HS Sultan, and MM.1S cell lines, in freshly derived patient MM cells (MM.As), MM cell lines resistant to dexamethasone (MM.1R), doxorubicin (DOX.40), mitoxantrone (MR.20), and mephalan (LR5). Importantly, after treatment with beta-lapachone, we observed no apoptosis in peripheral blood mononuclear cells in either quiescent or proliferative states, freshly isolated from healthy donors. In beta-lapachone treated ARH-77, cytochrome C was released from mitochondria to cytosol, and poly (ADP ribose) polymerase was cleaved, signature events of apoptosis. Finally, the apoptosis induced by beta-lapachone in MM cells was not blocked by either interleukin-6 or Bcl-2, which confer multidrug resistance in MM. CONCLUSIONS: Our results suggest potential therapeutic application of beta-lapachone against MM, particularly to overcome drug resistance in relapsed patients.

Antibiotics, Antineoplastic↗
PubMed1999

Potent inhibition of tumor survival in vivo by beta-lapachone plus taxol: combining drugs imposes different artificial checkpoints.

Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of beta-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after beta-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. beta-Lapachone causes cell-cycle delays in late G(1) and S phase, and taxol arrests cells at G(2)/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone "collisions" at cell-cycle checkpoints.

Animals↗
PubMed1999

Pronounced acute immunosuppression in vivo mediated by HIV Tat challenge.

HIV infection is accompanied by an early immune dysfunction limiting host control of virus and likely contributing to difficulties in achieving a successful vaccine against HIV. We report here that the HIV Tat protein is strongly immunosuppressive, both immediately after immunization of mice with soluble protein (sTat), and in seroconverting humans, and propose that Tat-induced suppression cripples immune surveillance to HIV infection. We show that macrophages are sensitive to sTat stimulation at concentrations 1,000-fold lower (500 pM) than T cells, and this stimulation is accompanied by the immunosuppressive induction of Fas ligand on the macrophage. T cell proliferative defects induced by sTat in vitro can be completely (at lower concentrations of sTat) or partially (at higher concentrations) reversed by antagonists to Fas/Fas ligand interaction. We further report a method to preserve immunogenicity while inactivating Tat immunosuppression through oxidation, which advances the use of oxidized Tat as a component of an anti-HIV vaccine. These observations define additional methods to study the immunosuppressive functions of sTat that now may be rapidly applied to primary isolates from individuals with differing clinical courses. Our findings have immediate relevance for vaccine development, by describing and supporting a strategy that includes inactivated sTat in a multicomponent, anti-HIV vaccine.

AIDS Vaccines↗
PubMed1999

Identification of a gene encoding a human oxysterol-binding protein-homologue: a potential general molecular marker for blood dissemination of solid tumors.

This study describes a new potential role in human cancer for a gene, HLM, isolated by differential display, that bears homology to an oxysterol-binding protein. A significant association between increased expression of HLM with metastatic disease was found. HLM mRNA levels were increased in circulating tumor cells in patients' peripheral blood and in primary human epithelial cells expressing the human papillomavirus16 E6 and E7 proteins. HLM mRNA was not detected in most normal human tissues, including peripheral blood and lymph node. These findings indicate that HLM may function as a potential marker for tumor dissemination.

Amino Acid Sequence↗
PubMed1999

Cancer and the cell cycle.

The purpose of this short review is to provide an overview of mammalian somatic cell cycle events and their controls. Cell cycle-related studies have been under way for only 5% of this millennium, yet since then nearly 20,000 references have appeared. This vast literature cannot be detailed here, nor can fundamental information obtained with other organisms such as yeast and Xenopus, or topics such as the abbreviated cell cycle in early embryonic cells. (General references include Murray and Hunt [1993] The cell cycle, an introduction. New York: Oxford University Press, and Denhardt [1999] In: The molecular basis of cell cycle and growth control. p 225-304. New York: John Wiley & Sons, Inc.) J. Cell Biochem. Suppls. 32/33:166-172, 1999.

Animals↗
PubMed1999

Association of K-ras mutations with p16 methylation in human colon cancer.

BACKGROUND & AIMS: K-ras mutations are early genetic changes in colon cancer. p16, a tumor-suppressor gene, is inactivated in neoplasms by mutation, deletion, or methylation. The aims of this study were to determine p16 methylation status and its possible association with K-ras mutations in human colon cancer. METHODS: DNA isolated from 8 colon cancer cell lines and 41 microdissected human colon tissue samples was analyzed. p16 methylation status was determined using two analytical methods. The level of p16 expression was determined by reverse-transcription polymerase chain reaction and Northern blot. K-ras mutations were determined by DNA sequence analysis. The DNA methyltransferase activity was determined by microassay. Parental and K-ras-transformed IEC-18 cells were used to determine the potential association between K-ras mutations and p16 methylation. RESULTS: Methylated p16 was found in 100% of colon cancer cell lines, 55% of colon cancers, 54% of adenomas, and 25% of transitional mucosa but not in normal colonic epithelium. Forty-five percent of cancers and 38% of adenomas showed both K-ras mutations and p16 methylation. Of 11 cancers and adenomas with K-ras mutation, 10 specimens showed methylated p16. In contrast, of 13 adenomas and cancers with wild-type K-ras, only 3 specimens showed methylated p16 (P = 0.001). Stable transformation of IEC-18 cells with K-ras increased the DNA methyltransferase activity, methylated the p16 gene, and suppressed the expression of p16. Treatment with a DNA methylation inhibitor (azadeoxycytidine) resulted in reexpression of p16 in K-ras-transformed IEC-18 cells, suggesting that the expression of p16 was suppressed by DNA methylation. CONCLUSIONS: p16 methylation occurs frequently in human colonic adenomas and cancers and is closely associated with K-ras mutations.

Azacitidine↗
PubMed1999

Evolution of transcriptional control from prokaryotic beginnings to eukaryotic complexities.

Mechanisms for regulating gene transcription became increasingly complex as organisms evolved. In prokaryotes the relatively simple mechanism of repression is based on a few proteins that bind to specific DNA sequences in a ligand-dependent fashion. In eukaryotes large complexes that include ligand binding proteins regulate transcription. Lower eukaryotes developed an additional level of control based on protein complexes that include modifying enzymes. The DNA/histone complex, in combination with gene-specific transcriptional factors, is the basis of gene regulation in eukaryotes. Higher eukaryotes took regulation a level further by methylating CpGs in promoter sequences of DNA, thereby allowing binding of histone deacetylases and inhibiting transcription. Finally, long-lasting "superrepression" provides another mechanism for coordinate transcriptional regulation of large blocks of genes.

Animals↗
PubMed1999

A strategy to identify genes associated with circulating solid tumor cell survival in peripheral blood.

Efforts in metastasis research have centered on the phenotypic and genetic differences between primary site and metastatic site tumors. However, genes that may be used as molecular markers of metastasis in circulating tumor cells remain unidentified. Genes regulating the dissemination and survival of solid tumor cells in the blood, as well as their adaptation to new environments, could be candidates for unique metastatic tumor markers. Differential display (DD) was conducted to compare the blood of tumor-free individuals with the blood of patients with lung, breast, and colon cancers. Twenty-one up-expressed genes in the tumor patient blood samples but none in the tumor-free donor blood samples were identified. Nine of these samples were isolated, amplified, and directly sequenced. A gene AB-1 homologous to a Bcl-2 family member, which might function as an apoptosis inhibitor, was identified. The overexpression of an apoptosis inhibitor in blood from patients with metastatic tumors might be correlated with the capability of solid tumor cells to survive in peripheral blood. This is the first demonstration of the usefulness of comparing control and patient blood samples by DD to find novel potential genetic markers identifying metastasis in the blood. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p313.html

Biomarkers, Tumor↗
PubMed1999

Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells.

BACKGROUND: There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C. MATERIALS AND METHODS: We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2. RESULTS: beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine. CONCLUSIONS: beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.

Antibiotics, Antineoplastic↗
PubMed1999

beta-lapachone induces cell cycle arrest and apoptosis in human colon cancer cells.

BACKGROUND: Human colon cancers have a high frequency of p53 mutations, and cancer cells expressing mutant p53 tend to be resistant to current chemo- and radiation therapy. It is thus important to find therapeutic agents that can inhibit colon cancer cells with altered p53 status. beta-Lapachone, a novel topoisomerase inhibitor, has been shown to induce cell death in human promyelocytic leukemia and prostate cancer cells through a p53-independent pathway. Here we examined the effects of beta-lapachone on human colon cancer cells. MATERIALS AND METHODS: Several human colon cancer cell lines, SW480, SW620, and DLD1, with mutant or defective p53, were used. The antiproliferative effects of beta-lapachone were assessed by colony formation assays, cell cycle analysis, and apoptosis analysis, including annexin V staining and DNA laddering analysis. The effects on cell cycle and apoptosis regulatory proteins were examined by immunoblotting. RESULTS: All three cell lines, SW480, SW620, and DLD1, were sensitive to beta-lapachone, with an IC(50) of 2 to 3 microM in colony formation assays, a finding similar to that previously reported for prostate cancer cells. However, these cells were arrested in different stages of S phase. At 24 hr post-treatment, beta-lapachone induced S-, late S/G2-, and early S-phase arrest in SW480, SW620, and DLD1 cells, respectively. The cell cycle alterations induced by beta-lapachone were congruous with changes in cell cycle regulatory proteins such as cyclin A, cyclin B1, cdc2, and cyclin D1. Moreover, beta-lapachone induced apoptosis, as demonstrated by annexin V staining, flow cytometric analysis of DNA content, and DNA laddering analysis. Furthermore, down-regulation of mutant p53 and induction of p27 in SW480 cells, and induction of pro-apoptotic protein Bax in DLD1 cells may be pertinent to the anti-proliferative and apoptotic effects of beta-lapachone on these cells. CONCLUSIONS: beta-Lapachone induced cell cycle arrest and apoptosis in human colon cancer cells through a p53-independent pathway. For human colon cancers, which often contain p53 mutations, beta-lapachone may prove to be a promising anticancer agent that can target cancer cells, especially those with mutant p53.

Antibiotics, Antineoplastic↗
PubMed1998

Calmodulin is essential for estrogen receptor interaction with its motif and activation of responsive promoter.

Calmodulin (CaM) has been reported to have affinity for the estrogen receptor (ER). Observations reported here reveal a direct physical interaction between purified CaM and ER. This direct ER-CaM interaction may be an initial event preceding the assembly of ER plus auxiliary proteins into the active ER complex with its DNA motif, the estrogen response element. We demonstrate that CaM is an integral component of this complex by using a system reconstituted from purified ER and nuclear extract from ER-negative breast cancer cells and also with ER-depleted nuclear extract of an ER-positive breast cancer cell line. Although CaM is essential for formation of this complex, it is not sufficient, suggesting roles also of auxiliary proteins. CaM also is functionally required for activation of an ER-responsive promoter, in the 17beta-estradiol-ER pathway of hormone action and regulation of 17beta-estradiol-responsive gene expression that is associated with proliferation of mammary epithelial cells.

Base Sequence↗
PubMed1998

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis.

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Base Sequence↗
PubMed1998

Inhibition of HIV-1 replication by combination of a novel inhibitor of TNF-alpha with AZT.

The small molecule S9a was derived from an established tumor necrosis factor-alpha (TNF-alpha) inhibitor (Canventol) by replacement of the isopropylidine group with a phenyl ring. S9a at 10 to 100 nM inhibited HIV production as potently as 3'-azido-3'-deoxythymidine (AZT), an inhibitor of viral reverse transcriptase. Furthermore, S9a and AZT in combination, at noncytoxic concentrations strongly inhibited HIV-1 replication that was more than additive and substantially prolonged the appearance of virus both in acutely infected CD4+ lymphocytes (SupT) in culture and in peripheral blood mononuclear cells (PBMCs) infected with a primary HIV-1 isolate. S9a inhibited TNF-alpha promoter-driven reporter gene activity. It was proposed that the mechanism of antiviral action of S9a was on the host cell, by blocking TNF-alpha transcription via a Tat-induced tar-independent loop, which decreases downstream NF-kappaB activation of HIV-1 long terminal repeat (LTR). S9a was superior to the first generation compound Canventol, which was superior to the natural compound sarcophytol A, demonstrating that further structure-based enhancement of potency of these compounds is feasible. This study suggests a therapeutic approach against AIDS by application of two drugs, one against a cellular and the other a viral target, which may provide an approach to the problem of frequent emergence of resistant variants to combinations of drugs that target only HIV genes.

Anti-HIV Agents↗
PubMed1998

Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin.

The maspin protein has tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion in vitro and tumor growth and metastasis in nude mice. Maspin is structurally a member of the serpin (serine protease inhibitors) superfamily but deviates somewhat from classical serpins. We find that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) are observed on plasminogen activation by sctPA. First, rMaspin(i) activates free sctPA. Second, it inhibits sctPA preactivated by poly-D-lysine. Third, rMaspin(i) exerts a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (< 0.5 microM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulates fibrinogen/gelatin-associated sctPA. rMaspin(i) acts specifically; it does not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA in vitro suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface.

Animals↗
PubMed1998

Differential display. A general protocol.

Characterization of regulated gene expression in eukaryotic cells is essential for studying cell growth and differentiation as well as for understanding the molecular mechanisms of diseases. Differential display was developed for such comparative studies by allowing a systematic and nonbiased screening for molecular differences at the level of mRNA expression between or among different cells or tissues. The essence of the method is to amplify messenger RNA 3' termini using a pair of anchored oligo-dT primer and a short primer with an arbitrary sequence. The amplified cDNAs labeled with radioisotope are then distributed on a denaturing polyacrylamide gel and visualized by autoradiography. Side-by-side comparison of mRNA species from two or more related samples allows identification of both up- and downregulation genes of interest. Some of the most recent improvements have been incorporated into this general protocol for differential display.

Base Sequence↗
PubMed1998

Apoptosis in non-proliferating cells: implications for viral infection and tumourigenesis.

To date much attention has been focused on regulation of apoptosis in proliferating cells. However, recent evidence shows that regulation of apoptosis in quiescent tissue plays an important role in homeostasis of the organism. This review examines the implications of apoptosis of quiescent cells for both tumourigenesis and viral infection such as HIV. In this article we propose a dual role for cellular activation in the homeostasis regulation. In this model cellular mitogens not only activate quiescent cells into the active cell cycle, but under certain conditions, loss of quiescence may result in apoptosis. The loss of quiescence-associated apoptosis may play a significant role in tumourigenesis and viral infections.

Journal Article↗