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H N Jayaram

Publications and source records attributed to H N Jayaram.

At least 19 recordsLinked to original sources
PubMed2004

Benzamide riboside induced mitochondrial mediated apoptosis in human lung cancer H520 cells.

Benzamide riboside (BR) is a novel anticancer agent exhibiting pronounced activity against several human tumor cell lines via the inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH), thereby restricting the biosynthesis of guanylates. Although it has been demonstrated that BR inhibits IMPDH and induces apoptosis, however, not much attention has been directed to the mechanism of apoptosis induction by this compound. The purpose of the present investigation was to investigate the mechanism of cytotoxicity induced by BR in human lung cancer cells. Non-small cell lung cancer [NSCLC] is the most prevalent type of lung cancer especially in India, and displays resistance to anticancer treatment. The results reveal that BR at a dose of 50 microM induces apoptosis in NSCLC H520 cells. This was ascertained by alteration in cellular morphology, TUNEL assay and flow cytometry. While Bax protein level was unaffected there was down regulation of anti-apoptotic Bcl-2 protein and up regulation of p53 as observed by Western blotting. Induction of apoptosis was accompanied by significant increase in caspase-3 activity. BR is a potent growth inhibitory pro-drug rationally synthesized to mimic NAD and inhibits PARP at high concentrations when assayed in permeabilized leukemic cells. Our observations showed that increased caspase-3 activity was accompanied by PARP cleavage. We also observed release of cytochrome c from mitochondria to the cytosol whereas no change was seen in the levels of apoptosis inducing factor (AIF). These findings indicate that BR induces apoptosis in H520 cells via the intrinsic mitochondrial pathway.

Antineoplastic Agents↗
PubMed2003

Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer.

AIMS: Folate receptors (FRs) mediate cellular uptake of folates in many cancer cells and in folate deficiency heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) mediates translational upregulation of FR in cultured cervical cancer cells. hnRNP-E1 can also interfere with human papillomavirus 16 (HPV-16) viral capsid protein synthesis (and thereby HPV proliferation) in vitro. This study aimed to evaluate prospectively the relevance of FR and hnRNP-E1 expression in the normal cervix, cervical dysplasia, and cancer. METHODS: Cervical tissues from 12 women with normal histology and 69 consecutive women with varying grades of cervical dysplasia and cancer were prospectively evaluated for immunohistochemical expression of FR, hnRNP-E1, proliferating cell nuclear antigen (PCNA), and HPV. There were 22 women with low grade squamous intraepithelial lesions (LGSIL), 22 with high grade squamous intraepithelial lesions (HGSIL), and 25 with invasive cervical carcinoma. RESULTS: Among normal subjects, 100% and 92% expressed hnRNP-E1 and FR, respectively. FR expression decreased from 91% in LGSIL to 68% and 64% in women with HGSIL and cancer, respectively. Similarly, hnRNP-E1 expression decreased from 86% in LGSIL to 68% and 40% in HGSIL and cancer, respectively. There was a highly significant positive correlation between the extent of FR and hnRNP-E1 expression, and an inverse correlation between HPV infection and hnRNP-E1 expression during progression of cervical dysplasia to cancer. CONCLUSION: These results are consistent with a hypothesis that reduced hnRNP-E1 expression may be permissive for HPV proliferation and progression to cervical cancer, and support the need for prospective longitudinal studies of hnRNP-E1 expression in HPV-16 infected women.

Biomarkers, Tumor↗
PubMed2002

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside.

A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5-10 microM range. Higher BR-concentrations (20 microM) predominantly induced necrosis which correlated with DNA strand breaks and subsequent depletion of ATP- and dATP levels. Replenishment of the ATP pool by addition of adenosine prevented necrosis and favoured apoptosis. This effect was not a pecularity of BR-treatment, but was reproduced with high concentrations of all trans-retinoic acid (120 microM) and cyanide (20 mM). Glucose was also capable to suppress necrosis and to favour apoptosis of HL-60 cells, which had been treated with necrotic doses of BR and cyanide. Apoptosis eliminates unwanted cells without affecting the microenvironment, whereas necrosis causes severe inflammation of surrounding tissues due to spillage of cell fluids into the peri-cellular space. Thus, the monitoring and maintenance of cellular energy pools during therapeutic drug treatment may help to minimize nonspecific side effects and to improve attempted drug effects.

Adenosine↗
PubMed2001

Metabolism and disposition of the novel antileukaemic drug, benzamide riboside, in the isolated perfused rat liver.

Benzamide riboside (BR) is a novel anticancer agent exhibiting pronounced activity against several human tumor cells, however, little is known about its biotransformation. To analyze for BR and its metabolites, livers of Wistar and mutant TR- rats were perfused with BR in a single pass system. In bile, native BR and its deamination product, benzene carboxylic acid riboside (BR-COOH) was quantified by HPLC. Total excretion of BR and BR-COOH into bile of Wistar rats was low (< 0.2%) whereas cumulative efflux of BR and its metabolite BR-COOH was high, representing 79% and 1.6% of infused BR, respectively. Biliary excretion of BR and BR-COOH in TR- rats, deficient in canalicular multispecific organic anion transporter, a membrane protein identical to MRP2 in tumor cells, was only slightly lower than in Wistar rats, indicating that BR and BR-COOH are non-substrates of MRP2. Experiments using rat hepatocytes incubated with BR did show a linear uptake of BR and a subsequent metabolism to BR-COOH that was largely excreted into the cellular supernatant. Examination of the cytotoxic activity against the human HL60 and K562 leukemia cells in a clonogenic assay demonstrated an IC50 of 619 microM and 1013 microM for BR-COOH compared to the IC50 of 0.21 microM and 0.46 microM for BR, suggesting the inertness of the metabolite. In summary, we found that deamination of BR to BR-COOH is the main metabolic pathway in rat liver. BR-COOH formation should also be considered in human liver during cancer therapy.

Animals↗
PubMed2001

Isolation and characterization of a folate receptor mRNA-binding trans-factor from human placenta. Evidence favoring identity with heterogeneous nuclear ribonucleoprotein E1.

The interaction of an 18-base cis-element in the 5'-untranslated region of human folate receptor (FR)-alpha mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539-25547). This trans-factor was isolated to apparent homogeneity as a 43- and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA cis-element in the 3'-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as alphaCP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.

Amino Acid Sequence↗
PubMed2001

Antitumor activity of benzamide riboside and its combination with cisplatin and staurosporine.

Benzamide riboside (BR), a new synthetic nucleoside analogue, has demonstrated a potent cytotoxic activity in murine leukemia in vitro. The purpose of the present investigation was to examine the antitumor activity of BR in mice bearing leukemia L1210. The results revealed that BR possesses a potent antitumor activity in vivo. It increases life-span of mice with leukemia. Synergistic cytotoxicity of BR with select DNA damaging agents, cisplatin (cis-Pt) and staurosporine (STP) was examined in MTT chemosensitivity assay, FACS analyses and apoptotic DNA fragmentation on L1210 cells in culture. A simultaneous treatment of leukemia L1210 cells with the combination of BR and STP resulted in synergistic cytotoxicity that correlated with increased apoptotic activity in those cells. On the other hand, treatment of L1210 cells with combination of BR and cis-Pt resulted in antagonistic cytotoxic effect. Finally, to elucidate the synergistic effect of BR and STP in inducing apoptosis, the attention was directed to the activation of cell death processes through various cell cycle signals. This is the first report describing in vivo antitumor activity of BR and its utilization in combination chemotherapy.

Animals↗
PubMed2000

Sensitizing human colon carcinoma HT-29 cells to cisplatin by cyclopentenylcytosine, in vitro and in vivo.

Cyclopentenylcytosine (CPEC) is cytotoxic to HT-29 cells in vitro and in vivo. Treatment with CPEC resulted in sensitizing HT-29 cells to cisplatin (CDDP), as evidenced by synergistic cytotoxicity. CPEC exhibits potent cytotoxicity to HT-29 cells in vitro, 2 and 24 h exposure providing an LC50 of 2.4 and 0.46 microM, respectively. Exposure of HT-29 cells to CDDP for 2 h resulted in an LC50 of 26 microM. Treatment of HT-29 cells with 1.0 or 1.25 microM CPEC and then incubating with CDDP showed synergistic cytotoxicity. Lesser synergy at very high concentrations of CPEC was demonstrated when HT-29 cells were first exposed to CDDP and then incubated with CPEC. Combination index calculations showed synergistic cytotoxicity in HT-29 cells when CPEC was combined with CDDP. Synergistic antitumor activity was demonstrable in vivo in mice transplanted with HT-29 tumor when treated with a combination of CPEC and CDDP without undue toxicity, since no excessive loss in mouse body weight or overt pathology was observed. CPEC had no influence on the total DNA adduct formation and CDDP did not affect the intracellular levels of CPEC or its metabolites, suggesting that enhanced CDDP cytotoxicity resulted from a step subsequent to excision of platinum-cross-linked DNA. These studies support a new approach for augmenting cytotoxic effect of CPEC with CDDP in treating human colon carcinoma.

Animals↗
PubMed2000

Synthesis, conformational analysis, and biological activity of C-thioribonucleosides related to tiazofurin.

The syntheses of furanthiofurin [5beta-D-(4'-thioribofuranosyl)furan-3-carboxamide, 1] and thiophenthiofurin [5beta-D-(4'-thioribofuranosyl)thiophene-3-carboxamide, 2], two C-thioribonucleoside analogues of tiazofurin, are described. Direct trifluoroacetic acid-catalyzed C-glycosylation of ethyl furan-3-carboxylate with 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha and beta anomers. Ethyl 5-(2,3,5-tri-O-benzyl)-beta-D-(4'-thioribofuranosyl)furan-3-carboxylate (6beta) was debenzylated and then converted into the corresponding amide (furanthiofurin) by reaction with ammonium hydroxide. A similar C-glycosylation of ethyl thiophene-3-carboxylate with 1,2,3,5-tetra-O-acetyl-4-thio-D-ribofuranose catalyzed by stannic chloride afforded an anomeric mixture of 2- and 5-glycosylated regioisomers. Deacetylation of ethyl 5-(2,3,5-tri-O-acetyl)-beta-D-(4'-thioribofuranosyl)thiophene-3-carboxylate (13beta) with methanolic ammonia and treatment of the ethyl ester with ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration were established by (1)H NMR spectroscopy. Thiophenthiofurin was found to be cytotoxic in vitro toward human myelogenous leukemia K562, albeit 39-fold less than thiophenfurin, while furanthiofurin proved to be inactive. K562 cells incubated with thiophenthiofurin resulted in inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) and an increase in IMP pools with a concurrent decrease in GTP levels. From computational studies it was deduced that, among the C-nucleoside analogues of tiazofurin, activity requires an electrophilic sulfur adjacent to the C-glycosidic bond and an energetically favorable conformer around chi = 0 degrees. Among these, the more constrained (least flexible) compounds (tiazofurin and thiophenfurin) are more active than the less constrained thiophenthiofurin. Those compounds which contain a nucleophilic oxygen in place of the thiazole or thiophene (oxazofurin, furanfurin, and furanthiofurin) show the least activity.

Antineoplastic Agents↗
PubMed1999

Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity.

Cervical carcinoma is an AIDS-defining illness. The expression of folate receptors (FRs) in cervical carcinoma (HeLa-IU1) cells was modulated by stable transduction of FR cDNA encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). Although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) FCS, [methyl-3H]thymidine incorporation into DNA was significantly increased in sense cells in 10% serum; therefore, the basis for this discrepancy was investigated. The activity of thymidine kinase (TK) was subsequently directly correlated with the extent of FR expression in single cell-derived clones of transduced cells. This elevated TK activity was not a result of recruitment of the salvage pathway based on the presence of adequate dTTP pools, normal thymidylate synthase (TS) activity, persistence of increased thymidine incorporation despite the exogenous provision of excess 5,10-methylene-tetrahydrofolate, and documentation of adequate folates in sense cells. The increase in TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as demonstrated by augmented phosphorylation of 3'-azido-3'-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT. Conversely, sense cells were highly resistant to methotrexate, but this was reversed by the addition of AZT. The direct correlation of FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression.

Antimetabolites, Antineoplastic↗
PubMed1999

Studies on the antitumor activity and biochemical actions of cyclopentenyl cytosine against human colon carcinoma HT-29 in vitro and in vivo.

Cyclopentenyl cytosine (CPEC) is cytotoxic to several tumor cell lines. CPEC inhibits CTP synthesis resulting in depletion of cytidylate pools. The aim of this study was to examine CPEC's cytotoxic and antitumor activity in vitro and in vivo against human colon carcinoma HT-29, and to relate its action on CTP synthesis. CPEC exhibits potent cytotoxicity in vitro to HT-29 cells with an LC50 (concentration that is lethal to the survival of 50% cell colonies) of 2.4 microM and 0.46 microM following 2 h and 24 h exposure, respectively. Incubation of cells with CPEC for 2 h resulted in a dose-dependent decrease in cytidylate pools. The in vivo antitumor activity of CPEC in athymic mice transplanted subcutaneously (s.c.) with 3 million HT-29 cells was examined. Antitumor activity of CPEC was elucidated in early-staged tumor, wherein CPEC (1.5 mg/kg, QD x 9 or 3 mg/kg, QOD x 9) was administered intraperitoneally (i.p.) 24 h after tumor implantation and it resulted in a significant reduction in tumor weight to 48% of control. The effect of CPEC on established solid tumors in vivo was examined in athymic mice transplanted s.c. 14 days earlier with HT-29 cells and treated i.p. with 1.5 mg/kg CPEC, QD x 5 for 4 courses, with a 10 day-interval between courses. This treatment resulted in a significant reduction in tumor weight (72%) in the treated group. HPLC analysis of HT-29 tumor obtained from mice after treatment with CPEC showed a depletion of the CTP concentration reaching a nadir at 8 h. In conclusion, the present studies demonstrate potent antitumor activity of CPEC against freshly transplanted and established human colon carcinoma which can be corroborated with the drug's biochemical actions.

Adenosine Triphosphate↗
PubMed1999

Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells.

One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10 - 20 microM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive c-Myc expression.

Adenocarcinoma↗
PubMed1999

Consequences of IMP dehydrogenase inhibition, and its relationship to cancer and apoptosis.

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.

Antineoplastic Agents↗
PubMed1998

Isosteric analogues of nicotinamide adenine dinucleotide derived from furanfurin, thiophenfurin, and selenophenfurin as mammalian inosine monophosphate dehydrogenase (type I and II) inhibitors.

Dinucleotides TFAD (6), FFAD (7), and SFAD (8), isosteric NAD analogues derived, respectively, from C-nucleosides 5-beta-d-ribofuranosylthiophene-3-carboxamide (thiophenfurin, 1), 5-beta-d-ribofuranosylfuran-3-carboxamide (furanfurin, 2), and 5-beta-d-ribofuranosylselenophene-3-carboxamide (selenophenfurin, 5), were synthesized as human inosine monophosphate dehydrogenase (IMPDH) type I and II inhibitors. The synthesis was carried out by imidazole-catalyzed coupling of the 5'-monophosphate of 1, 2, and 5 with AMP. These dinucleotides, which are also analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD), the active metabolites of the oncolytic C-nucleosides 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) and 2-beta-D-ribofuranosylselenazole-4-carboxamide (selenazofurin), were evaluated for their inhibitory potency against recombinant human IMPDH type I and II. The order of inhibitory potency found was SAD > SFAD = TFAD = TAD >> FFAD for both enzyme isoforms. No significant difference was found in inhibition of IMPDH type I and II.

Enzyme Inhibitors↗
PubMed1998

Staurosporine enhanced benzamide riboside-induced apoptosis in human multidrug-resistant promyelocytic leukemia cells (HL-60/VCR) in vitro.

The inosine monophosphate (IMP) dehydrogenase inhibitor benzamide riboside (BR) induced apoptosis (detected with the aid of flow cytometric identification of cells with sub-G0 DNA content and increased side angle light scatter) equally or slightly more intensively in the multidrug-resistant human promyelocytic leukemia cell line (HL-60/VCR: MDR-1 gene, Pgp positive) in comparison with the parental drug sensitive HL-60 cells. Staurosporine alone induced relatively low level of apoptosis in parental HL-60 cells but higher level (approximately 35%) of apoptosis in multidrug-resistant HL-60/VCR cells after 24 hour induction. The combination of benzamide riboside and staurosporine induced in both drug-sensitive and drug-resistant HL-60 cells a marked proportion of apoptotic cells already after short (6 hour) induction (more than 30% of apoptotic cells).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Synthesis, structure, and antiproliferative activity of selenophenfurin, an inosine 5'-monophosphate dehydrogenase inhibitor analogue of selenazofurin.

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.

Animals↗
PubMed1996

P-selectin-mediated attachment of small cell lung carcinoma to endothelial cells.

Small cell lung carcinoma (SCLC) frequently metastasizes early in the course of the disease. P-selectin (P-sel), a cell adhesion molecule expressed on activated platelets and endothelial cells (EC), has previously been demonstrated to mediate binding of platelets to SCLC. We hypothesized that P-sel facilitates attachment of SCLC to EC, acting as an important factor in SCLC metastasis. To test this hypothesis, attachment of H82 cells (SCLC cell line) to EC was quantified. Attachment of H82 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated EC was increased compared with control EC. Increased attachment of H82 cells to EC was apparent after 10 min of TPA activation, reached a peak after 30 min, and returned to baseline after 120 min of exposure. The TPA-induced increase in H82 cell attachment to EC was inhibited by addition of anti-P-sel antibodies but not by addition of anti-E-selectin antibodies. The TPA-induced increase in H82 cell attachment was likely mediated by activation of EC protein kinase C (PKC). Pretreatment of the EC with PKC inhibitors effectively blocked the TPA-mediated increase in H82 cell attachment. In addition, prolonged exposure of EC to TPA resulted in decreased expression of the PKC-alpha and PKC-epsilon isoforms. These data indicate for the first time that attachment of SCLC to activated EC appears to be mediated by increased expression of P-sel on the EC surface, which may result from activation of specific isoforms of PKC.

Animals↗
PubMed1996

Studies on the mechanism of action of 1-beta-D-arabinofuranosyl-5-azacytosine (fazarabine) in mammalian lymphoblasts.

Fazarabine has shown activity in the panel of 60 cultured human tumor lines of the National Cancer Institute. COMPARE analyses relating correlation coefficients of other anticancer drugs with those of fazarabine suggest that this agent operates through a similar mode of action to that of cytarabine. Studies have been carried out both in culture and in vivo to examine the mechanism of action of fazarabine in P388 murine and Molt-4 human lymphoblasts. Authentic fazarabine nucleotide standards were prepared by chemical and enzymatic methods and characterized on HPLC by comparison to related pyrimidine nucleoside-5'-phosphates as well as by enzymatic digestion. Fazarabine inhibited the incorporation of labeled thymidine into DNA without influencing the synthesis of RNA or protein. Deoxycytidine overcomes this inhibition of DNA synthesis and also prevents the cytotoxicity of the drug to lymphoblasts, probably by competing for fazarabine uptake and metabolism. Fazarabine was rapidly phosphorylated in both cell lines; in P388 cells it was incorporated into DNA, where it continued to undergo the same type of ring opening and degradation as the free nucleoside. Alkaline elution studies demonstrated that exposure to the agent resulted in the formation of alkaline labile sites. Fazarabine also inhibited the methylation of deoxycytidine residues in DNA, but this effect was less pronounced than that produced by 5-azacytidine. Taken together, these studies suggest that fazarabine probably acts by arresting the synthesis and/or altering the structural integrity or functional competence of DNA.

Animals↗