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Biomedical subjects

D Samid

Publications and source records attributed to D Samid.

At least 55 records · Page 3Linked to original sources
PubMed1994

Selective activity of phenylacetate against malignant gliomas: resemblance to fetal brain damage in phenylketonuria.

Phenylacetate, a deaminated metabolite of phenylalanine, has been implicated in damage to immature brain in phenylketonuria. Because primary brain tumors are highly reminiscent of the immature central nervous system, these neoplasms should be equally vulnerable. We show here that sodium phenylacetate can induce cytostasis and reversal of malignant properties of cultured human glioblastoma cells, when used at pharmacological concentrations that are well tolerated by children and adults. Treated tumor cells exhibited biochemical alterations similar to those observed in phenylketonuria-like conditions, including selective decline in de novo cholesterol synthesis from mevalonate. Because gliomas, but not mature normal brain cells, are highly dependent on mevalonate for production of sterols and isoprenoids vital for cell growth, sodium phenylacetate would be expected to affect tumor growth in vivo while sparing normal tissues. Systemic treatment of rats bearing intracranial gliomas resulted in significant tumor suppression with no apparent toxicity to the host. The data indicate that phenylacetate, acting through inhibition of protein prenylation and other mechanisms, may offer a safe and effective novel approach to treatment of malignant gliomas and perhaps other neoplasms as well.

Animals↗
PubMed1994

Upregulation of tumor necrosis factor-alpha production by retrovirally transduced human tumor-infiltrating lymphocytes using trans-retinoic acid.

The ability of retinoic acid (RA) to upregulate gene expression in human tumor-infiltrating lymphocytes (TIL) transduced with a Moloney murine leukemia virus containing the cDNA encoding tumor necrosis factor (TNF) has been studied. TNF production was increased approximately twofold after treatment with RA. This increase was dose dependent and corresponded to a rise in the level of LTR-driven mRNA measured by Northern analysis. RA did not appreciably increase transcription by the SV40 promoter or increase endogenous TNF production. The effect lasted for 3-6 days following withdrawal of RA. These studies indicate that RA can upregulate LTR-driven gene expression in TIL cells bearing retroviral vectors and may thus be of use in studies of the gene therapy of cancer.

Gene Expression↗
PubMed1994

In vitro antitumor effect of hydroxyurea on hormone-refractory prostate cancer cells and its potentiation by phenylbutyrate.

Previous clinical trials have suggested that hydroxyurea may possess some activity against prostate cancer. The in vitro antiproliferative activity of hydroxyurea was evaluated in three hormone-refractory prostate cancer cell lines, PC-3, DU-145 and PC-3M. Fifty-percent inhibition of growth in all three cell lines required prolonged (120 h) exposure to hydroxyurea at a concentration of approximately 100 microM. Using pharmacokinetic data obtained during the course of a clinical trial of hydroxyurea, we simulated a dosing regimen that would sustain plasma drug concentrations above 100 microM for 120 h (1 g loading dose, followed by 500 mg every 6 h for 5 days in a 70 kg man). Since this dosing regimen is likely to generate an unacceptable degree of myelosuppression, in vitro combination studies were conducted with hydroxyurea and phenylbutyrate, a new differentiating agent with no myelosuppressive effects. These studies resulted in a reduction of the hydroxyurea concentration necessary for 50% growth inhibition (50 microM of hydroxyurea plus 0.5 mM of phenylbutyrate). A regimen designed to achieve that hydroxyurea concentration (400 mg loading dose, followed by 200 mg every 6 h for 5 days) should be clinically achievable. Based on these results, this combination deserves further evaluation in patients with stage D prostate cancer.

Cell Division↗
PubMed1994

Differentiation of cultured human melanoma cells induced by the aromatic fatty acids phenylacetate and phenylbutyrate.

The increasing incidence of melanoma and the poor responsiveness of disseminated disease to conventional treatments call for the development of new therapeutic approaches. Phenylacetate, a nontoxic differentiation inducer, can suppress the growth of other neuroectodermal tumors, i.e., gliomas, in laboratory models and in humans. This finding led us to explore the efficacy of phenylacetate and related aromatic fatty acids in melanoma. Phenylacetate and phenylbutyrate were found to a) induce selective cytostasis and maturation of cultured human melanoma cells, b) modulate the expression of genes implicated in tumor metastasis (type IV collagenase and tissue inhibitor of metalloproteinases-2) and immunogenicity (HLA class I); and c) enhance the efficacy of other agents of clinical interest, including retinoids, interferon-alpha, suramin, and 5-aza-2'-deoxycytidine. Reflecting on the phenotypic heterogeneity of melanoma, the degree of biologic alterations induced by phenylacetate/phenylbutyrate varied significantly among the tumor cell lines tested. Although losing invasive capacity and tumorigenicity in athymic mice, poorly differentiated cells exhibited only a marginal change in morphology, remained amelanotic, and resumed growth after treatment was discontinued. By contrast, treatment of melanoma cells that were in a more advanced stage of maturation resulted in profound alterations in cell growth, morphology, and pigmentation consistent with terminal differentiation. The in vitro antitumor activity was observed with nontoxic, pharmacologic concentrations of phenylacetate and phenylbutyrate, suggesting potential clinical use of these drugs in the treatment of melanomas.

Antineoplastic Agents↗
PubMed1993

Development of a novel spontaneous metastasis model of human osteosarcoma transplanted orthotopically into bone of athymic mice.

There is a pressing need for in vivo models in which potential antitumor agents can be tested for their ability to inhibit the growth and metastatic spread of human sarcomas. A recent advance in this regard has been the development of a v-Ki-ras-oncogene-transformed human osteosarcoma cell line (KRIB) that efficiently colonizes the lungs of athymic nude mice when cells (1 x 10(5)) are administered by i.v. injection. In the present study, we have utilized this cell line to develop a spontaneous metastasis model in which a small number of tumor cells are injected into the tibial bones of athymic mice. When as few as 1000 KRIB cells are orthotopically implanted into the tibial bones of nude mice, bone tumors, which are radiographically and histologically similar to primary human osteosarcoma, develop within 4 weeks. Furthermore, as in the human disease, cells from these primary tumors subsequently seed the animals' lungs, resulting in reproducible and quantifiable pulmonary metastasis evident both upon gross inspection of the lungs and histologically 6 weeks after tumor inoculation. Surgical amputation of the tumor inoculation site up to 2 weeks after tumor injection prevents pulmonary metastasis, indicating that substantial local (tibial) growth and invasion of the primary tumor for at least 2 weeks is required for subsequent metastasis. Implantation of s.c. 5000 KRIB cells fails to produce local or metastatic tumors. We anticipate that this model will prove to be a powerful tool with which to study the mechanisms of human osteosarcoma growth and pulmonary metastasis, and to assess the efficacy of promising therapeutic agents.

Animals↗
PubMed1993

Enhanced fetal hemoglobin production by phenylacetate and 4-phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia.

In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

Anemia, Sickle Cell↗
PubMed1993

Increased radioresistance of EJras-transformed human osteosarcoma cells and its modulation by lovastatin, an inhibitor of p21ras isoprenylation.

Alterations in ras oncogene expression have been associated with increased cellular resistance to ionizing radiation. As an extension of studies with murine cell models, we have now explored the radioresponses of human osteosarcoma (HOS) sub-clones that differ in their EJras expression. Quantitative analysis revealed a tight correlation between the amounts of ras-encoded mRNA and p21 produced, and the degree of cell radioresistance. Interestingly, treatment of the ras-transformed cells with lovastatin, an inhibitor of p21ras post-translational processing via the mevalonate pathway, markedly decreased their radioresistance. Under the experimental conditions used, lovastatin prevented the membrane association, but not the biosynthesis, of p21. The decline in radiation resistance following lovastatin treatment could not be attributed to perturbation of cholesterol metabolism or to non-specific cell-cycle effects. In agreement, lovastatin did not alter the radiation responses of control HOS cells that do not express EJras, or those with an activated met oncogene. The results indicate that elevation in ras gene expression can lead to increased radioresistance of human tumor cells. It appears, however, that p21ras membrane localization is critical for maintenance of the radioresistant phenotype, thus providing a target for pharmacological intervention.

Animals↗
PubMed1993

Differences in radiation-induced micronuclei yields of human cells: influence of ras gene expression and protein localization.

Expression of ras has been correlated with increased intrinsic resistance to ionizing radiation. In this study we show that increased EJras expression in human cells is associated with a decrease in the frequency of radiation-induced micronuclei. The experimental system consisted of human osteosarcoma-derived cell lines which quantitatively vary in their EJras expression. There was a dose-dependent relationship between radiation dose and micronuclei formation in all cell lines tested. Human osteosarcoma cells, in which the ras level was undetectable, had the highest frequency of micronuclei production at all radiation doses tested. At 4 Gy the most radioresistant cells exhibited a 41.5 +/- 5% decrease in the production of micronuclei concomitant with high ras expression in comparison with the relatively radiosensitive parental cell line. Cells expressing a low amount of EJras demonstrated a 23 +/- 3% decrease in micronuclei induction compared with parental cells. Treatment of cells with lovastatin, an inhibitor of ras-encoded p21ras post-translational processing via the mevalonate pathway, markedly decreased the yield of micronuclei formation in cells transfected with ras; the drug had no effect on radiation-induced micronuclei formation in parental cells. The use of the in vitro micronuclei assay has provided a convenient way to visualize differences in the genotoxic damage induced by ionizing radiation in cells which express different amount of EJras. The results indicate that elevation of ras expression in human cells can lead to a decrease in the number of radiation-induced micronuclei formed and that this relationship is dependent on membrane association of ras-encoded p21.

Cell Line↗
PubMed1993

Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione.

Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.

3T3 Cells↗
PubMed1993

Selective growth arrest and phenotypic reversion of prostate cancer cells in vitro by nontoxic pharmacological concentrations of phenylacetate.

Differentiation therapy may provide an alternative for treatment of cancers that do not respond to cytotoxic chemotherapy or hormonal manipulations. This hypothesis led us to evaluate the effect of a nontoxic differentiation inducer, sodium phenylacetate (NaPA), on hormone-refractory prostate cancer, the second most common cause of cancer deaths in men. NaPA treatment of androgen-independent PC3 and DU145 prostate cell lines, like that of hormone-responsive LNCaP cultures, resulted in dose-dependent inhibition of cell proliferation. Similar treatments were not significantly inhibitory to replicating normal endothelial cells and skin fibroblasts. In addition to the selective cytostatic effect, NaPA induced reversion of the prostatic cells to a nonmalignant phenotype, evidenced by their reduced invasiveness and loss of tumorigenicity in athymic mice. Phenotypic reversion was accompanied by alterations in gene expression, including selective reduction in tumor growth factor-beta 2 mRNA levels and increased amounts of class I major histocompatibility complex HLA transcripts. Furthermore, there was a decrease in tumor-associated proteolysis mediated by urokinase plasminogen activator, a molecular marker of disease progression in humans. When tumor cells were treated with NaPA together with suramin, a drug with demonstrable activity in patients, there was complete abrogation of cell growth under conditions in which each treatment alone produced only a partial effect. The in vitro antineoplastic activity was observed with drug concentrations that have been achieved in humans with no significant toxicities, suggesting that PA, used alone or in combination with other antitumor agents, warrants evaluation in the treatment of advanced prostatic cancer.

Animals↗
PubMed1993

Review: molecular biology of transgenic animals.

The amazing pace of discovery within the field of molecular biology has expanded the understanding of as well as the potential for genetic engineering in laboratory and domestic animals. Recently, specific techniques have been perfected, including recombinant DNA, genetic cloning, analysis of gene expression, and the sequencing of nucleic acids and proteins. These molecular techniques combined with methods allowing transfer of the recombinant genes into living cells and fertilized eggs advent the production of transgenic mice and other animals, the application of which is unlimited.

Animals↗
PubMed1992

Induction of erythroid differentiation and fetal hemoglobin production in human leukemic cells treated with phenylacetate.

There is considerable interest in identifying nontoxic differentiation inducers for the treatment of various malignant and nonmalignant blood disorders, including inborn beta-chain hemoglobinopathies. Using the human leukemic K562 cell line as a model, we explored the efficacy of phenylacetate, an amino acid derivative with a low toxicity index when administered to humans. Treatment of K562 cultures with pharmacologically attainable concentrations of phenylacetate resulted in erythroid differentiation, evident by the reduced growth rate and increased hemoglobin production. The effect was time- and dose-dependent, further augmented by glutamine starvation (phenylacetate is known to deplete circulating glutamine in vivo), and reversible upon cessation of treatment. Molecular analysis showed that phenylacetate induced gamma globin gene expression with subsequent accumulation of the fetal form of hemoglobin (HbF). Interestingly, the addition of phenylacetate to antitumor agents of clinical interest, eg, hydroxyurea and 5-azacytidine, caused superinduction of HbF biosynthesis. The results suggest that phenylacetate, used alone or in combination with other drugs, might offer a safe and effective new approach to treatment of some hematopoietic neoplasms and severe hemoglobinopathies.

Azacitidine↗
PubMed1992

Phenylacetate: a novel nontoxic inducer of tumor cell differentiation.

Sodium phenylacetate was found to affect the growth and differentiation of tumor cells in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of promyelocytic leukemia HL-60 cells resulted in the rapid decline of myc oncogene expression followed by growth arrest and granulocyte differentiation. Phenylacetate also induced highly efficient adipocyte conversion in immortalized mesenchymal C3H 10T1/2 cultures; yet, unlike the differentiating chemotherapeutic drug 5-aza-2'-deoxycytidine, phenylacetate did not cause neoplastic transformation in these susceptible cells. The results indicate that phenylacetate is both effective in inducing tumor cell maturation and free of cytotoxic and carcinogenic effects, a combination that warrants attention to its potential use in cancer intervention.

Adipose Tissue↗
PubMed1992

Changes in gene expression by 193- and 248-nm excimer laser radiation in cultured human fibroblasts.

Tissue ablation by ultraviolet excimer lasers results in exposure of viable cells to subablative doses of radiation. To understand the potential biological consequences better, we have studied changes in gene expression in cultured human skin fibroblasts exposed to either 193- or 248-nm laser light. Northern blot analyses revealed that both treatments up-regulate a common set of genes, including interstitial collagenase, tissue inhibitor of metalloprotease, metallothionein, and the proto-oncogene c-fos. Dose-response and kinetic studies of collagenase induction by 193-nm radiation showed a maximal effect with 60 J/m2 and at approximately 24 h. The induction was still persistent 96 h later. In addition to the commonly affected genes, known to be activated also by conventional UV light (254 nm) and tumor-promoting phorbol esters, other genes were found to be selectively induced by the 193-nm radiation. The heat-shock hsp70 mRNA, undetectable in controls and in cultures irradiated at 248 nm, was transiently induced 8 h after exposure to 193-nm radiation. Furthermore, a selective up-regulation of collagen type I expression was observed. The results indicate that the 193- and 248-nm radiations by excimer lasers elicit specific and different cellular responses, in addition to an overlapping pathway of gene activation common also to UV radiation by germicidal lamps. The laser-induced genes could serve as molecular markers in evaluating cell injury in situ.

Collagenases↗
PubMed1991

Increased sensitivity of nontumorigenic fibroblasts expressing ras or myc oncogenes to malignant transformation induced by 5-aza-2'-deoxycytidine.

The hypomethylating chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC) has been shown to induce cell differentiation in some systems, while promoting neoplastic transformation in others. Using both in vitro and in vivo models, we have explored the relationship between oncogene expression and the susceptibility of cells to malignant transformation by 5AzadC. The study involved several nontumorigenic subclones of NIH3T3 fibroblasts, including cells transfected with deregulated c-myc, as well as phenotypic revertants expressing v-Ki-ras or long terminal repeat-activated c-Ha-ras. Transient 5AzadC treatment of the oncogene-bearing cell lines was associated with a rapid and efficient neoplastic transformation. In some cases, over 50% of the cell population exhibited loss of contact inhibition of growth within 1 week of treatment. The transformants were capable of forming s.c. tumors and experimental lung metastases in recipient nude mice. In contrast, 5AzadC failed to induce malignant properties in control 3T3 cultures transfected with the bacterial neor gene; rather, treatment of these cells was associated with differentiation into adipocytes and myotubes. The differential response to 5AzadC was also observed in vivo, in mice first inoculated s.c. with the premalignant cells and then treated with 5AzadC 24 h later. In agreement with the in vitro model, tumor development in mice correlated with the presence of cells with activated ras or myc oncogenes. Cytidine analogs that do not inhibit DNA methylation (i.e., 6-azacytidine and 1-beta-D-arabinofuranosyl cytosine) had no effect on cell phenotype. The results indicate that exposure of cells to 5AzadC can lead to tumor progression both in vitro and in vivo and suggest that preexisting alterations in oncogene expression may facilitate the evolution of cancerous growth induced by hypomethylating agents.

Animals↗
PubMed1991

DNA damage in cultured human skin fibroblasts exposed to excimer laser radiation.

Ultraviolet excimer lasers are being considered for use in a variety of refractive and therapeutic procedures, the long-term biologic consequences of which are unknown. The effect of sublethal doses of 193-nm laser radiation on cellular DNA was examined in cultured human skin fibroblasts. In contrast to 248 nm, treatments with the 193-nm laser radiation below 70 J/m2 did not cause significant pyrimidine dimer formation in the skin cells. This was indicated by the lack of excision repair activities (unscheduled DNA synthesis assay), and further demonstrated by direct analysis of pyrimidine dimers in DNA from irradiated cells. However, a low level of unscheduled DNA synthesis could be detected following irradiation at 193 nm with 70 J/m2. Both the 193-nm and 248-nm radiation were able to induce chromosomal aberrations, as indicated by a micronucleus assay. A dose-dependent increase in micronuclei frequency was observed 48 and 72 h after laser irradiation. These results indicate that exposure of actively replicating human skin fibroblasts to sublethal doses of either 193- or 248-nm laser radiation can result in genotoxicity.

Cell Survival↗
PubMed1991

Interferon in combination with antitumourigenic phenyl derivatives: potentiation of IFN alpha activity in-vitro.

Any attempt to eradicate the heterogeneous cell population of a tumour mass would require the use of appropriate combination treatment protocols. The antitumour effects of interferon alpha (IFN alpha) in combination with AS2-1, the hydrolysis product of 3-phenylacetyl-amino-2,6-piperidinedione, were examined using several human tumour cell lines as a model. These included the malignant melanoma A375, adenocarcinoma of the prostate PC3 (hormone-insensitive bone metastasis), and the erythroleukaemia line K562. AS2-1 suppressed tumour growth through non-toxic mechanisms, with 1 mg/ml causing approximately 50% inhibition of the melanoma and prostate tumour cell proliferation. By contrast, primary normal human skin fibroblasts were significantly less sensitive to the antiproliferative effect of AS2-1. Suppression of tumour growth was seen also with AS2-1 treatment of the erythroleukaemia K562; in these cultures the drug also induced dose-dependent differentiation, as indicated by the increased haemoglobin production. Interestingly, addition of low doses of IFN alpha markedly enhanced the antitumour and differentiating effects observed with AS2-1. Treatment with 200-300 IU/ml of IFN (which caused about 20% inhibition of growth) together with 1 mg/ml of AS2-1 resulted in over 80% inhibition of the melanoma and prostate cancer cell proliferation, suggesting a synergistic activity of the two agents. This was substantiated by quantitative analysis of the differentiation induced in K562 erythroleukaemia. It appears, therefore, that IFN alpha and AS2-1 may act through synergistic mechanisms to effectively inhibit tumour growth and promote differentiation in a variety of human malignant cell lines.

Cell Division↗