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P M Schwartz

Publications and source records attributed to P M Schwartz.

At least 19 recordsLinked to original sources
PubMed1999

Expression and localization of thymidine phosphorylase/platelet-derived endothelial cell growth factor in skin and cutaneous tumors.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TPase/PD-ECGF) is a catabolic enzyme that has been shown to be chemotactic for endothelial cells in vitro and angiogenic in vivo. TPase/PD-ECGF expression is increased in a variety of tumors. In the skin, TPase is active in normal keratinocytes in vitro and in vivo. Our objective was to study the expression and localization of TPase/PD-ECGF by immunohistochemical analysis in normal skin and cutaneous tumors and to correlate this information with enzymatic activity of TPase. TPase/PD-ECGF expression was observed in keratinocytes with intense staining of the infundibulum of hair follicles but no staining of hair bulbs. Expression localized primarily to the nucleus of keratinocytes in the basal layer but was more intense and cytoplasrmic in suprabasal keratinocytes. Increased expression of TPase/PD-ECGF in differentiated cells was confirmed by in vitro studies of TPase activity. In cutaneous tumors, there was positive staining for TPase/ PD-ECGF in squamous cell carcinomas (10/10), eccrine poromas (3/4), eccrine syringomas (4/4), trichoepitheliomas (1/3), and tumors of the follicular infundibulum (2/3) and melanomas (5/8). There was no staining of any intradermal nevi (0/2), basal cell carcinomas (0/10) or Merkel cell carcinoma (0/1). We conclude TPase/PD-ECGF is found throughout the epidermis and its expression increases with differentiation of keratinocytes. In cutaneous tumors, expression of TPase/PD-ECGF may be linked to the cell of origin of the tumor as well as the tumor's degree of differentiation.

Antibody Specificity↗
PubMed1998

Beta-L-1,3-dioxolane-cytidine: a novel nucleoside that inhibits proliferation and induces differentiation of keratinocytes in vitro.

beta-L-1,3-Dioxolane-cytidine (L-(-)-OddC) is a novel L-nucleoside, and its antitumor activity is under investigation in clinical trials. To evaluate the potential of L-(-)-OddC for treating hyperproliferative diseases of the skin, we examined its activity in human keratinocytes in vitro. The dose of L-(-)-OddC that inhibited the rate of proliferation of keratinocytes by 50% was 50 nM. L-(-)-OddC was about as cytotoxic as 9-beta-D-arabinofuranosylcytosine but was about 1,000 time more potent than 3'-azidothymidine. L-(-)-OddC caused irreversible growth arrest and induced differentiation of keratinocytes. L-(-)-OddC altered morphology, increased the cell size of keratinocytes and increased the expression of involucrin. These data suggest that L-(-)-OddC may have potential as a therapeutic agent against hyperproliferative skin diseases.

Antimetabolites, Antineoplastic↗
PubMed1997

Keratinocyte gene therapy for adenosine deaminase deficiency: a model approach for inherited metabolic disorders.

Disorders in which there is toxic buildup of circulating substrate may be treated by furnishing an enzyme reservoir capable of metabolically processing the excess substrate. The epidermal keratinocyte is a potential site for such a reservoir. In this study, we explore the capacity of genetically modified keratinocytes to metabolize extracellular substrate in a culture model that resembles in vivo epidermal architecture. Keratinocytes from adenosine deaminase (ADA)-deficient patients were transduced with a retroviral vector encoding the human ADA gene and the capacity of this tissue to deaminate deoxyadenosine (dAdo) in vitro was measured. The results show that at a substrate concentration of 10 microM, ADA-corrected keratinocytes deaminate dAdo at a rate of 0.38 nmol/min.10(6) cells. These results indicate that keratinocytes process extracellular substrate at rates that suggest complete substrate conversion in a single pass. This study provides a strong indication that the epidermis, the largest and most accessible tissue of the body, is a valuable site for designing clinically relevant gene therapies.

Adenosine Deaminase↗
PubMed1997

Abnormal cerebellar development and foliation in BDNF-/- mice reveals a role for neurotrophins in CNS patterning.

While target-derived neurotrophins are required for the survival of developing neurons in the PNS, the functions of neurotrophins in the CNS are unclear. Mice with a targeted gene deletion of brain-derived neurotrophic factor (BDNF) exhibit a wide-based gait. Consistent with this behavioral evidence of cerebellar dysfunction, there is increased death of granule cells, stunted growth of Purkinje cell dendrites, impaired formation of horizontal layers, and defects in the rostral-caudal foliation pattern. These abnormalities are accompanied by decreased Trk activation in granule and Purkinje cells of mutant animals, indicating that both cell types are direct targets for BDNF. These data suggest that BDNF acts as an anterograde or an autocrine-paracrine factor to regulate survival and morphologic differentiation of developing CNS neurons, and thereby affects neural patterning.

Animals↗
PubMed1995

Keratinocytes differentiate in response to inhibitors of deoxyribonucleotide synthesis.

We previously reported that methotrexate (MTX) caused an irreversible inhibition of growth and induced terminal differentiation of human keratinocytes. These effects of methotrexate were prevented by thymidine and thus, were attributed to depletion of thymine deoxyribonucleotides. The purpose of the research reported in this paper was to determine whether differentiation was induced by the general class of agents which are known to interfere with synthesis or utilization of deoxyribonucleotides. Agents examined included 5-fluorouracil, 5-bromodeoxyuridine, hydroxyurea, high-dose thymidine, aphidicolin, and AG#85, a newly reported thymidylate synthase inhibitor. All these agents increased the expression of involucrin and increased the amount of cornified envelope protein at doses that inhibited proliferation by > 75%. We demonstrated, however, that in our cell culture system not all conditions producing inhibition of proliferation induced differentiation; withdrawal of growth factors and supplemental amino acids inhibited proliferation but did not increase involucrin expression or production of cornified envelope protein.

Cell Differentiation↗
PubMed1994

Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.

Activating Transcription Factor 3↗
PubMed1992

Methotrexate induces differentiation of human keratinocytes.

Terminal differentiation is a key element in the maintenance of tissue homeostasis in the epidermis. We show here that methotrexate (MTX) induces differentiation of human epidermal keratinocytes in vitro. MTX inhibits proliferation of keratinocytes and also induces several markers of differentiation: a change in cell morphology, a marked increase in cell size, an increase in the proportion of cells that express involucrin, and an increase in the amount of cornified envelope protein. These effects of MTX are dose- and exposure-time-dependent and become irreversible after 24 hr, approximately one population doubling time. These effects of MTX cannot be attributed to cytotoxicity since keratinocytes not only remain viable but also actively synthesize proteins. MTX causes reproducible changes in the SDS/PAGE profiles of newly synthesized proteins and, in particular, increases the amount of involucrin synthesis. Thymidine completely prevents these effects of MTX, suggesting that they are caused by a depletion of thymine deoxyribonucleotides. The effect of MTX on keratinocytes may provide a model for studying the relationship between deoxyribonucleotide metabolism and differentiation in normal cells. In addition, the ability of MTX to induce differentiation in keratinocytes suggests a mechanism to explain its therapeutic action in psoriasis.

Cell Differentiation↗
PubMed1991

Thymidine salvage changes with differentiation in human keratinocytes in vitro.

We compared the capacity of proliferating and differentiating keratinocytes to salvage and catabolize extracellular thymidine. Both populations of cells catabolized thymidine to thymine and possessed thymidine phosphorylase activity. As keratinocytes differentiate, thymidine phosphorylase activity ultimately increased twofold. In contrast, proliferating and differentiating keratinocytes differed markedly in their capacity to salvage extracellular thymidine. Proliferating keratinocytes readily salvaged extracellular thymidine to form nucleotides, whereas differentiating cells rapidly lost this capacity. The inability of differentiating cells to form nucleotides from thymidine was not attributed to reduced availability of thymidine due to catabolism but rather was the result of the rapid loss of thymidine kinase activity. As keratinocytes differentiate in suspension culture, they lose 41% of thymidine kinase activity in 8 h and over 90% of activity in 12 h. Our data indicate that loss of capacity to salvage extracellular thymidine for synthesis of nucleotides closely parallels the onset of differentiation in keratinocytes.

Cell Differentiation↗
PubMed1991

Antiviral activity of 5-iodo-2'-deoxyuridine and related drugs in human keratinocytes infected in vitro with herpes simplex virus type 1.

5-Iodo-2'-deoxyuridine (IUDR) is a potent topical antiviral agent in experimental animals but is less active in man for treating cutaneous viral infections. We have shown here that IUDR is 5 times less active in human keratinocytes infected in vitro with herpes simplex virus type 1 than in guinea pig embryo cells infected in culture. To account, in part, for this difference in activity of IUDR, we measured the capacity of these different cultures to catabolize and thus inactivate the drug. IUDR is catabolized by thymidine phosphorylase; activity of this enzyme was very high in human keratinocytes in vitro but was very low in guinea pig embryo cells. The antiviral activity of IUDR in human keratinocytes, however, was not increased by inhibiting thymidine phosphorylase; inhibiting thymidine phosphorylase apparently increased the availability of thymidine that would compete with IUDR and, indeed, the activity of IUDR in infected cells was reduced by addition of thymidine to the medium. These data indicate that the catabolism of IUDR and related analogs alters antiviral activity in human keratinocytes.

Animals↗
PubMed1989

Dipyridamole potentiates the growth-inhibitory action of methotrexate and 5-fluorouracil in human keratinocytes in vitro.

Human keratinocytes transport extracellular thymidine across the plasma membrane and incorporate it into DNA. Data presented here show that dipyridamole, a well-known inhibitor of facilitated diffusion of nucleosides, blocks the transport of thymidine into human keratinocytes in vitro. Dipyridamole (1.0 microM) inhibited the transport of 3H-thymidine (0.2 microM) into intracellular material by 75% and its subsequent salvage and incorporation into DNA by 48%. Dipyridamole (1 microM) did not affect the growth of keratinocytes in vitro but did potentiate the growth inhibition caused by methotrexate (MTX) or 5-fluorouracil (5-FU). The growth of keratinocytes exposed to 0.1 microM MTX for 8 d was inhibited by 32%. However, in combination with a noninhibitory concentration of dipyridamole (1 microM), this concentration of MTX (0.01 microM) inhibited the growth of keratinocytes by 93%. Thymidine in culture medium reversed the cytotoxicity of MTX. However, in the presence of dipyridamole, thymidine in the culture medium did not reverse the action of MTX. The synergistic interaction between MTX and dipyridamole was also observed with 5-FU and dipyridamole. 5-FU (0.5 microM) inhibited cell growth by 30% but in combination with dipyridamole (1 microM), inhibited cell growth by 86%. These data are consistent with the theory that inhibiting thymidine salvage by blocking transport of extracellular thymidine potentiates the growth inhibitory action of inhibitors of de novo pyrimidine biosynthesis in human keratinocytes. Combination chemotherapy, such as methotrexate plus dipyridamole, might be efficacious in the treatment of hyperproliferative diseases of the epidermis.

Cell Division↗
PubMed1988

Human keratinocytes catabolize thymidine.

Human neonatal foreskin keratinocytes incorporate exogenous thymidine into DNA and proliferate in vitro even after reaching confluence. Keratinocytes also catabolize thymidine, as reported for the first time below. Stratified cultures of keratinocytes reduced the amount of thymidine in the medium by more than 90% within 2 to 4 h. Consequently, the rate of incorporation of thymidine (0.2 microM, 4 microCi/ml) into DNA was linear for no more than 2 h. Linear incorporation of thymidine into DNA for at least 12 h could be achieved by continual addition of fresh radioactive thymidine to the culture medium. Different tissues have widely differing abilities to catabolize thymidine. Cutaneous catabolism of thymidine shows striking species differences. Soluble extracts from human neonatal foreskin and adult skin, as well as from cultivated human keratinocytes, actively catabolize thymidine. Soluble extracts of skin from mouse, rabbit, or guinea pig do not catabolize thymidine. Extracts from cultivated human fibroblasts and melanocytes have little or no ability to catabolize thymidine. Catabolism of thymidine by keratinocytes has important implications for the use of [3H]thymidine in studies of keratinocyte proliferation and for the use of thymidine analogs in therapy of cutaneous disease.

Cells, Cultured↗
PubMed1988

Local and systemic implications of thymidine catabolism by human keratinocytes.

The human epidermis possesses a very active thymidine phosphorylase with the capacity to catabolize all of the thymidine available to the epidermis from the circulation and from the degradation of DNA in terminally differentiating keratinocytes. This high capacity of keratinocytes to catabolize thymidine could affect local levels of thymidine within the epidermis for DNA synthesis and could contribute to the regulation of the concentration of thymidine in the systemic circulation. Further work is needed to delineate the physiologic role of this keratinocyte enzyme. A practical consequence of the activity of epidermal thymidine phosphorylase is the role it may play in limiting the clinical efficacy of certain thymidine analogs, an important class of antiviral agents.

Cells, Cultured↗
PubMed1985

Role of uridine phosphorylase in the anabolism of 5-fluorouracil.

The activities of enzymes responsible for activating 5-fluorouracil (FUra) to 5-fluorouridine-5'-monophosphate (FUMP) were compared in normal and tumor tissues of rodents to assess the potential capacity of uridine phosphorylase to anabolize FUra to the nucleoside in the presence of ribose-1-phosphate (R-1-P). The activity of the alternative pathway to FUMP with a pyrimidine phosphoribosyltransferase [FUra + 1-pyrophosphoribosyl-5-phosphate (PPRP)] was approximately 15 to 17 nmoles/mg protein/hr in bone marrow from mice and rats and ranged from 28 to 47 nmoles/mg protein/hr in tumor tissues. Uridine phosphorylase [measured as the formation of 5-fluorouridine (FUrd) from FUra and R-1-P] was 35-230 nmoles/mg/hr in bone marrow and in two FUra-sensitive solid tumors, colon tumor No. 38 in mice and RPMI colon tumor in rats; the activity of uridine phosphorylase from L5178Y ascites leukemic cells was notably lower, 8 nmoles/mg/hr. Levels of uridine kinase ranged from 55 to 187 nmoles/mg protein/hr. Thus, the activities of the enzymes of the two-step FUra activating pathway were high compared to the PPRP-dependent activity in all tissues except L5178Y; also, the FUra-sensitive tumors yielded extracts with 1.5 to 6.5 times greater enzyme activity than the corresponding activity in bone marrow. Uridine phosphorylase was partially purified from rat liver, RPMI rat tumor and colon tumor No. 38; the apparent Km of FUra averaged 50 microM, almost 9-fold lower than that of uracil, and the apparent Km of R-1-P for condensation with FUra was 33 microM. The tissue concentration of R-1-P was greater than 70 microM in kidney and liver of rodents and somewhat less in spleen. Colon tumor No. 38 and RPMI colon tumor had 12 and 20 microM R-1-P, respectively, but these low values may reflect low tumor viability. The high levels of uridine phosphorylase and uridine kinase activities in normal tissues and even higher levels in tissues from FUra-sensitive tumors, as well as the sufficient concentration of R-1-P relative to its kinetic constant, suggest that FUra metabolism by the two-step pathway to FUMP may be a significant factor in the activity and selectivity of FUra.

Animals↗
PubMed1985

The effect of intrauterine PCB exposure on visual recognition memory.

Adverse neonatal outcomes have been associated with intrauterine exposure to polychlorinated biphenyls (PCBs). In a follow-up study of exposed and nonexposed infants, 123 infants tested at birth were administered Fagan's test of visual recognition memory at 7 months. 2 measures of prenatal PCB exposure, cord serum PCB level and maternal report of contaminated fish consumption, both predicted less preference for a novel stimulus. Preference for novelty decreased in a dose-dependent fashion with increasing levels of prenatal PCB exposure. Postnatal exposure from nursing was not related to visual recognition memory. The relation between prenatal exposure and visual recognition was not mediated by the neonatal deficits, suggesting that intrauterine PCB exposure may have a delayed effect on central nervous system (CNS) functioning.

Adolescent↗
PubMed1985

Altered plasma kinetics of 5-FU at high dosage in rat and man.

To provide a model for pharmacokinetic studies in patients administered high iv doses of 5-FU after allopurinol or other modifiers of toxicity, rats were given radioactive 5-FU as an iv bolus. At a dose of 50 mg/m2 only 16% of circulating radioactivity was identified as 5-FU after 30 minutes, whereas greater than 90% of radioactivity was 5-FU when rats were given a 1800-mg/m2 dose. This 36-fold increase in 5-FU dose was associated with a greater than 150-fold increase in the 30-minute plasma concentration of 5-FU (0.02-3.3 mM). In patients, it was possible to increase the maximum tolerated dose of 5-FU given as a weekly rapid iv bolus from 800 to 1700 mg/m2 when a 3-day course of allopurinol (900 mg/day orally) preceded 5-FU administration. A dose-dependent increase in the apparent half-life of 5-FU in the plasma was observed and plasma concentrations at 30 minutes were as high as 500 microM after a 1600-mg/m2 dose of the drug.

Animals↗