Biomedical subjects

D J Waxman

Publications and source records attributed to D J Waxman.

At least 19 recordsLinked to original sources

PubMed2006

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy.

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.

Animals↗

PubMed2002

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer.

Tissue-like structures of cells organized in vitro have a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1 transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.

Adenoviridae↗

PubMed2001

Inhibitory cross-talk between STAT5b and liver nuclear factor HNF3beta: impact on the regulation of growth hormone pulse-stimulated, male-specific liver cytochrome P-450 gene expression.

STAT5b is repeatedly activated in rodent liver by the male pattern of intermittent plasma growth hormone (GH) stimulation and is required to maintain the GH pulse-regulated, male-specific pattern of liver gene expression. We presently investigate the interactions between STAT5b and hepatocyte-enriched nuclear factors (HNFs) that contribute to regulation of GH pulse-inducible, male-specific liver cytochrome P-450 (CYP) genes. STAT5 binding sites were identified in the 5'-flank of the adult male-expressed genes CYP2A2 (nucleotides -2255 to -2247), CYP4A2 (nucleotides -1872 to -1864), and CYP2C11 (nucleotides -1150 to -1142). STAT5-DNA complexes were formed by each CYP sequence with nuclear extract from GH pulse-activated male, but not female, rat liver. The CYP2C11 STAT5 site, which is flanked by HNF3 consensus sequences, conferred STAT5b-inducible reporter gene activity in GH-treated HepG2 cells. trans-Activation of the intact CYP2C11 promoter (1.8-kilobase 5'-flank) was strongly induced by the liver nuclear factors HNF1alpha and HNF3beta but, unexpectedly, was inhibited by GH-activated STAT5b. This STAT5b inhibitory effect could be reversed by HNF1alpha and reflects a functional antagonism between STAT5b and HNF3beta, as evidenced by the inhibition of HNF3beta DNA binding and transcriptional activity by STAT5b. HNF3beta, in turn, inhibited STAT5b by a novel mechanism that leads to suppression of GH-inducible STAT5b tyrosine phosphorylation, DNA binding activity, and transcriptional activity. The potential for GH-activated STAT5b to stimulate male-specific liver CYP expression can thus be modulated by HNF3beta, highlighting the complex interrelationship between STAT5b and liver transcription factors controlling expression of GH-regulated CYP genes.

Animals↗

PubMed2001

Frequent, moderate-dose cyclophosphamide administration improves the efficacy of cytochrome P-450/cytochrome P-450 reductase-based cancer gene therapy.

Transduction of tumor cells with a cyclophosphamide (CPA)-activating cytochrome P-450 (P450) gene provides the capacity for localized prodrug activation and greatly sensitizes solid tumors to CPA treatment in vivo. The therapeutic impact of this P450-based cancer gene therapy strategy can be substantially enhanced by cotransduction of P450 reductase, a rate-limiting component of P450-dependent intratumoral CPA activation. The present study examined the possibility of further improving P450/P450 reductase-based gene therapy by using a novel schedule of CPA administration, involving repeated CPA injection every 6 days and previously shown to have an antiangiogenic component. 9L gliosarcoma cells transduced with the CPA-activating enzyme couple P450 2B6/P450 reductase and grown s.c. in immunodeficient severe combined immunodeficient (scid) mice were repeatedly challenged with 140 mg/kg CPA every 6 days. Full tumor regression leading to eradication of six of eight tumors was observed when the tumor size at the time of initial drug treatment was approximately 400 mm(3) (approximately 1.5% of body weight). Little or no overt toxicity of the repeated CPA treatment regimen was observed. The same CPA schedule was much less effective in inducing regression of 9L tumors that were not transduced with P450/P450 reductase. Repeated CPA treatment of mice bearing large, late-stage P450/P450 reductase-transduced tumors (approximately 9-16% of body weight) resulted in major (> or =95%) regression in 15 of 16 tumors, with tumor eradication observed in 2 cases. Although CPA resistance was found to emerge in the population of P450/P450 reductase-transduced tumors, this resistance primarily involved a loss of expression of the transduced P450 and/or P450 reductase gene, rather than development of intrinsic cellular resistance to the activated form of CPA. These findings demonstrate that repeated CPA treatment on a 6 day schedule can be highly effective when combined with P450/P450 reductase gene therapy and suggest that repeated transduction of tumors with prodrug-activation genes may be necessary to achieve tumor eradication and a sustained therapeutic response.

Animals↗

PubMed2001

An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.

Hepatic hydroxylation is an essential step in the metabolism and excretion of bile acids and is necessary to avoid pathologic conditions such as cholestasis and liver damage. In this report, we demonstrate that the human xenobiotic receptor SXR (steroid and xenobiotic receptor) and its rodent homolog PXR (pregnane X receptor) serve as functional bile acid receptors in both cultured cells and animals. In particular, the secondary bile acid derivative lithocholic acid (LCA) is highly hepatotoxic and, as we show here, a metabolic substrate for CYP3A hydroxylation. By using combinations of knockout and transgenic animals, we show that activation of SXR/PXR is necessary and sufficient to both induce CYP3A enzymes and confer resistance to toxicity by LCA, as well as other xenotoxicants such as tribromoethanol and zoxazolamine. Therefore, we establish SXR and PXR as bile acid receptors and a role for the xenobiotic response in the detoxification of bile acids.

Animals↗

PubMed2001

Modulation of cyclophosphamide-based cytochrome P450 gene therapy using liver P450 inhibitors.

The sensitivity of tumors to cyclophosphamide (CPA) and other anticancer prodrugs can be substantially enhanced by transduction of tumors with a prodrug-activating mammalian cytochrome P450 (CYP) enzyme in combination with the flavoenzyme P450 reductase. This gene therapy strategy provides for intratumoral prodrug activation, but is also associated with a high level of hepatic prodrug activation, which reduces the extent of intratumoral prodrug activation and contributes to systemic drug toxicity. To address this issue, five P450 inhibitors were tested for their ability to block liver CYP2C-catalyzed CPA activation selectively, i.e., without inhibiting the corresponding intratumoral activation of CPA catalyzed by a transduced CYP2B enzyme. In vitro studies revealed that the P450 inhibitors 1-aminobenzotriazole and DDEP were preferentially inhibitory toward CYP2C-dependent liver microsomal CPA activation, whereas the P450 inhibitor SKF-525A inhibited CYP2C- and CYP2B-dependent CPA activation without P450 form selectivity. By contrast, the P450 inhibitors chloramphenicol and metyrapone preferentially inhibited CYP2B-dependent CPA activation. Rat pharmacokinetic studies confirmed the inhibitory action of these compounds in vivo, with up to a 4-fold decrease in C(max) and a 7-fold increase in apparent half-life of the activated CPA metabolite, 4-hydroxy-CPA, seen in the case of 1-aminobenzotriazole. Although the rate of hepatic CPA activation could thus be decreased substantially by P450 inhibitor treatment, the net extent of hepatic CPA activation was only modestly decreased, as judged by plasma area-under-the-curve values for 4-hydroxy-CPA. Moreover, P450 inhibitor treatment did not decrease CPA's host toxicity and did not enhance the tumor growth delay response to CPA in rats bearing CYP2B1-transduced gliosarcomas. These findings are discussed in the context of P450-based gene therapy strategies and ongoing efforts to enhance anticancer drug activity by increasing the exposure of P450-expressing tumors to the P450-activated prodrug CPA.

Animals↗

PubMed2001

Transcriptional induction of hepatic NADPH: cytochrome P450 oxidoreductase by thyroid hormone.

Studies were carried out to elucidate the mechanism whereby thyroid hormone (T3) induces NADPH:cytochrome P450 oxidoreductase (P450R) mRNA in rat liver in vivo. Northern blot analysis revealed that T3 treatment increases unspliced liver nuclear P450R RNA 4-fold within 8 h and that this induction precedes the induction of mature, cytoplasmic P450R RNA. Unspliced nuclear P450R RNA was suppressed below basal levels 24 h after T3 treatment, despite the continued presence of elevated circulating T3 levels. To determine whether the T3-stimulated increase in nuclear P450R RNA reflects an increase in P450R transcription initiation, nuclear run-on transcription assays were carried out. T3 induced a 6- to 8-fold increase in P450R transcription rate within 12 h, sufficient to account for the observed increase in nuclear P450R precursor RNA, followed by a decrease back to basal transcription levels at 24 h, consistent with the nuclear RNA profile. Similar transcriptional increases were observed in nuclear run-on transcription studies using hybridization probes corresponding to nine different fragments of the P450R gene, spanning exon 2 to exon 16. Thus, P450R transcription initiation, not transcription elongation, is the T3-regulated event. Similar results were obtained during short (5 min) compared with long (45 min) nuclear run-on transcription assays, suggesting that changes in nuclear RNA processing or regulated degradation do not contribute to the overall RNA induction. This finding was confirmed by the ability of the RNA polymerase inhibitor actinomycin D, administered in vivo, to block T3 induction of P450R transcriptional activity. We conclude that P450R transcription, rather than nuclear RNA processing or mRNA stabilization, is the primary mechanism whereby T3 induces hepatic P450R mRNA.

Animals↗

PubMed2001

Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells.

Cyclophosphamide (CPA), a widely used oxazaphosphorine anti-cancer prodrug, is inactive until it is metabolized by cytochrome P450 to yield phosphoramide mustard and acrolein, which alkylate DNA and proteins, respectively. Tumor cells transduced with the human cytochrome P450 gene CYP2B6 are greatly sensitized to CPA, however, the pathway of CPA-induced cell death is unknown. The present study investigates the cytotoxic events induced by CPA in 9L gliosarcoma cells retrovirally transduced with CYP2B6, or induced in wild-type 9L cells treated with mafosfamide (MFA) or 4-hydroperoxyifosfamide (4OOH-IFA), chemically activated forms of CPA and its isomer ifosfamide. CPA and MFA were both shown to effect tumor cell death by stimulating apoptosis, as evidenced by the induction of plasma membrane blebbing, DNA fragmentation, and cleavage of the caspase 3 and caspase 7 substrate poly(ADP-ribose) polymerase (PARP) in drug-treated cells. Caspase 9 was identified as the regulatory upstream caspase activated in 9L cells treated with CPA, MFA, or 4OOH-IFA, implicating the mitochondrial apoptotic pathway in oxazaphosphorine-induced tumor cell death. Correspondingly, expression of the mitochondrial proapoptotic factor Bax enhanced caspase 9 activation, plasma membrane blebbing, and drug-induced cytotoxicity. Conversely, overexpression of the mitochondrial antiapoptotic factor Bcl-2 blocked caspase 9 activation, leading to an inhibition of drug-induced plasma membrane permeability and blebbing, terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity, PARP cleavage, Annexin V positivity, and drug-induced cell death. Although Bcl-2 thus blocked the cytotoxic effects of activated CPA, it did not inhibit the drug's cytostatic effects. CPA induced S-phase cell cycle arrest followed by conversion to an apoptotic pre-G1 state in wild-type 9L cells; by contrast, Bcl-2-expressing 9L cells accumulated in G2/M in response to CPA treatment. Intratumoral expression of Bcl-2 and related family members, including both apoptotic and antiapoptotic factors, is thus an important determinant of the responsiveness of tumor cells to CPA and ifosfamide, both in the context of conventional chemotherapy and in patients sensitized to these oxazaphosphorine drugs by the use of cytochrome P450-based gene therapy.

Animals↗

PubMed2001

Temporal relationship between the sexually dimorphic spontaneous GH secretory profiles and hepatic STAT5 activity.

STAT5 transduces transcriptional responses to GH in liver and other tissues and is proposed to mediate the sexually dimorphic effects of plasma GH secretory profiles on rodent liver gene expression. Previous studies have suggested that STAT5 undergoes repeated activation in direct response to successive GH pulses in adult male rats, with STAT5 activation being desensitized in females by their more persistent pattern of GH exposure. These findings, however, were based on in vitro studies or single blood samples analyzed for GH in vivo. In view of the highly pulsatile nature of rat GH secretion, we presently examined these hypotheses by concurrent monitoring of spontaneous GH secretory profiles and hepatic STAT5 activity in conscious, free-moving adult male and female rats. Rats were killed at times associated with spontaneous peaks or troughs of the GH rhythm; livers were removed and analyzed for STAT5 DNA-binding activity. In males, liver STAT5 activity was highest during the initial phase (15-60 min) of a GH secretory episode (mean +/- SE relative STAT5 activity = 86.5 +/- 11.4; plasma GH = 146.7 +/- 22.4 ng/ml) and was significantly lower (P < 0.01) during the downswing of a pulse, 45-75 min after the GH peak (STAT5 = 26.1 +/- 1.7; GH = 33.3 +/- 13.1 ng/ml), consistent with a time-dependent down-regulation of GH signaling to STAT5. The lowest STAT5 activity was observed during the subsequent GH trough period (STAT5 = 3.6 +/- 1.1; GH = 2.6 +/- 0.1 ng/ml). In females, liver STAT5 activity was significantly lower (P < 0.05) than peak male levels during the initial phase of a GH secretory burst (STAT5 = 35.1 +/- 15.9; GH = 68.1 +/- 31.6 ng/ml) although similar to that of males during a plasma GH nadir (STAT5 = 11.0 +/- 2.6; GH = 8.4 +/- 2.2 ng/ml). We conclude that: 1) liver STAT5 is repeatedly activated by successive, spontaneous GH secretory episodes in intact adult male rats at approximately 3- to 3.5-h intervals; 2) time-dependent down-regulation of GH signaling to hepatic STAT5 in vivo begins by 45 min after GH peak stimulation; and 3) the lower level of liver STAT5 activation seen in adult female rats, compared with males, is a consequence of the sex-dependent differences in GH secretory patterns that characterize these animals (i.e. lower-amplitude GH pulses and lack of prolonged interpulse nadir of GH in the feminine, compared with masculine profile).

Animals↗

PubMed2001

STAT5b is required for GH-induced liver IGF-I gene expression.

Although the increased expression of Igf-I in liver in response to GH is well characterized, the intracellular signaling pathways that mediate this effect have not been identified. Intracellular signaling molecules belonging to the Janus kinase-signal transducer and activator of transcription 5b (JAK2-STAT5b) pathway are activated by GH and have previously been shown to be required for sexually dimorphic body growth and the expression of liver cytochrome P450 proteins known to be regulated by the gender-specific temporal patterns of pituitary GH secretion. Here, we evaluate the role of STAT5b in GH activation of Igf-I by monitoring the induction of Igf-I mRNA in livers of wild-type and Stat5b(-/-)mice stimulated with exogenous pulses of GH. GH induced the expression of liver Igf-I mRNA in hypophysectomized male wild-type, but not in hypophysectomized male Stat5b(-/-) mice, although the Stat5b(-/-) mice exhibit both normal liver GH receptor expression and strong GH induction of Cytokine-inducible SH2 protein (Cis), which is believed to contribute to the down-regulation of GH-induced liver STAT5b signaling. Thus, STAT5b plays an important and specific role in liver Igf-I gene expression.

Animals↗

PubMed2001

Serine phosphorylation of GH-activated signal transducer and activator of transcription 5a (STAT5a) and STAT5b: impact on STAT5 transcriptional activity.

Signal transducer and activator of transcription 5b (STAT5b), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5b DNA-binding and transcriptional activities, the effect of STAT5b serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5b serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b without inhibiting STAT5b nuclear translocation. Evaluation of the functional consequences of STAT5b serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5b Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity was not diminished. No decrease in GH-stimulated reporter activity was seen with the corresponding STAT5a-Ser725Ala mutant; however, a decrease in reporter activity occurred when the second established STAT5a serine phosphorylation site, serine 779, was additionally mutated to alanine. Unexpectedly, STAT5a-Ser725,779Ala and STAT5b-Ser730Ala displayed approximately 2-fold higher GH- or PRL-stimulated transcriptional activity compared with wild-type STAT5b when assayed using an intact beta-casein promoter-luciferase reporter. Finally, STAT5b-stimulated gene transcription was abolished in cells treated with H7, but in a manner unrelated to the inhibitory effects of H7 on STAT5b Ser730 phosphorylation. These findings suggest that the effects of STAT5b and STAT5a serine phosphorylation on STAT-stimulated gene transcription can be modulated by promoter context. Moreover, in the case of STAT5a, phosphorylation of serine 779, but not serine 725, may serve to regulate target gene transcriptional activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2001

P450 enzyme expression patterns in the NCI human tumor cell line panel.

Cytochrome P450 (P450) enzyme expression patterns were determined for a panel of 60 human tumor cell lines, representing nine tumor tissue types, used by the National Cancer Institute (NCI) Anticancer Drug Screening Program. All 60 tumor cell lines displayed significant P450 activity, as well as P450 reductase activity, as determined using the general P450 substrate 7-benzyloxyresorufin. Cell line-specific P450 enzyme patterns were observed using three other P450 substrates, 7-ethoxycoumarin, coumarin, and 7-ethoxyresorufin, each of which was metabolized at a low rate. Using a pattern-matching computer program, COMPARE, correlative relationships were investigated between the arrays of P450 activities and the patterns of cytotoxicity exhibited by a large group of anticancer agents of proven or potential clinical utility. Significant negative correlations between the patterns of P450-dependent 7-benzyloxyresorufin metabolism activity and cell line chemosensitivity were observed for 10 standard anticancer agents (including 6 alkylating agents) and 55 investigational compounds, suggesting a role for P450 metabolism in the inactivation of these agents. Negative correlations between 7-ethoxycoumarin O-deethylation and cell line chemosensitivity to a group of topoisomerase inhibitors were also seen, again suggesting P450-dependent drug inactivation. P450 enzyme profiling may thus aid in interpreting the patterns of drug sensitivity and resistance in the NCI tumor cell panel, and may facilitate the identification of anticancer agents whose activity can be altered via cytochrome P450 metabolism.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2000

Role of the cytokine-inducible SH2 protein CIS in desensitization of STAT5b signaling by continuous growth hormone.

Growth hormone (GH)-inducible suppressors of cytokine signaling (SOCS/CIS proteins) inhibit GH receptor (GHR) signaling to STAT5b via phosphotyrosine-dependent binding interactions with the tyrosine kinase JAK2 (SOCS-1) and/or the cytoplasmic tail of GHR (CIS and SOCS-3). Presently, we investigate the mechanism of CIS inhibition and CIS's role in down-regulating GHR-JAK2 signaling to STAT5b in cells exposed to GH continuously. CIS is shown to inhibit GHR-JAK2 signaling by two distinct mechanisms: by a partial inhibition that is decreased at elevated STAT5b levels and may involve competition between CIS and STAT5b for common GHR cytoplasmic tail phosphotyrosine-binding sites; and by a time-dependent inhibition, not seen with SOCS-1 or SOCS-3, that involves proteasome action. Investigation of the latter mechanism revealed that GH stimulates degradation of CIS, but not SOCS-3. The proteasome inhibitor MG132 blocked this protein degradation and also blocked the inhibitory action of CIS, but not that of SOCS-1 or SOCS-3, on STAT5b signaling. Proteasome-dependent degradation of CIS, most likely in the form of a (GHR-JAK2)-CIS complex, is therefore proposed to be an important step in the time-dependent CIS inhibition mechanism. Finally, the down-regulation of GHR-JAK2 signaling to STAT5b seen in continuous GH-treated cells could be prevented by treatment of cells with the proteasome inhibitor MG132 or by expression of CIS-R107K, a selective, dominant-negative inhibitor of CIS activity. These findings lead us to propose that the cytokine signaling inhibitor CIS is a key mediator of the STAT5b desensitization response seen in cells and tissues exposed to GH chronically, such as adult female rat liver.

Animals↗

PubMed2000

Synergistic action of hepatocyte nuclear factors 3 and 6 on CYP2C12 gene expression and suppression by growth hormone-activated STAT5b. Proposed model for female specific expression of CYP2C12 in adult rat liver.

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription through its sex-dependent temporal pattern of pituitary hormone secretion. CYP2C12 encodes a female-specific rat liver P450 steroid hydroxylase whose expression is activated by continuous GH stimulation of hepatocytes. Presently, we investigated the role of liver-enriched and GH-regulated transcription factors in the activation of CYP2C12 gene expression in GH-stimulated liver cells. Transcription of a CYP2C12 promoter-luciferase reporter gene in transfected HepG2 cells was activated 15-40-fold by the liver-enriched hepatocyte nuclear factor (HNF) 3 alpha, HNF3 beta, and HNF6. Synergistic interactions leading to an approximately 300-fold activation of the promoter by HNF3 beta in combination with HNF6 were observed. 5'-Deletion analysis localized the HNF6 response to a single 5'-proximal 96-nucleotide segment. By contrast, the stimulatory effects of HNF3 alpha and HNF3 beta were attributable to five distinct regions within the 1.6-kilobase CYP2C12 proximal promoter. GH activation of the signal transducer and transcriptional activator STAT5b, which proceeds efficiently in male but not female rat liver, inhibited CYP2C12 promoter activation by HNF3 beta and HNF6, despite the absence of a classical STAT5-binding site. The female-specific pattern of CYP2C12 expression is thus proposed to reflect the positive synergistic action in female liver of liver-enriched and GH-regulated transcription factors, such as HNF3 beta and HNF6, coupled with a dominant inhibitory effect of GH-activated STAT5b that is manifest in males.

Animals↗

PubMed2000

Combination of the bioreductive drug tirapazamine with the chemotherapeutic prodrug cyclophosphamide for P450/P450-reductase-based cancer gene therapy.

Tirapazamine (TPZ) is a bioreductive drug that exhibits greatly enhanced cytotoxicity in hypoxic tumor cells, which are frequently radiation-resistant and chemoresistant. TPZ exhibits particularly good activity when combined with alkylating agents such as cyclophosphamide (CPA). The present study examines the potential of combining TPZ with CPA in a cytochrome P450-based prodrug activation gene therapy strategy. Recombinant retroviruses were used to transduce 9L gliosarcoma cells with the genes encoding P450 2B6 and NADPH-P450 reductase. Intratumoral coexpression of P450 2B6 with P450 reductase sensitized 9L tumor cells to CPA equally well under normoxic (19.6% O2) and hypoxic (1% O2) conditions. The P450 2B6/P450 reductase combination also sensitized 9L tumor cells to TPZ under both culture conditions. Interestingly, bystander cytotoxic effects were observed for both CPA and TPZ under hypoxia. Furthermore, TPZ exerted a striking growth-inhibitory effect on CPA-treated 9L/2B6/P450 reductase cells under both normoxia and hypoxia, which suggests the utility of this drug combination for P450-based gene therapy. To evaluate this possibility, 9L tumor cells were transduced in culture with P450 2B6 and P450 reductase and grown as solid tumors in severe combined immune deficient mice in vivo. Although these tumors showed little response to TPZ treatment alone, tumor growth was significantly delayed, by up to approximately four doubling times, when TPZ was combined with CPA. Some toxicity from the drug combination was apparent, however, as indicated by body weight profiles. These findings suggest the potential benefit of incorporating TPZ, and perhaps other bioreductive drugs, into a P450/P450 reductase-based gene therapy strategy for cancer treatment.

Animals↗

PubMed2000

Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide.

The anticancer alkylating agents cyclophosphamide (CPA) and ifosfamide (IFA) are prodrugs that undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic (N-dechloroethylated) metabolites. Whereas the human liver microsomal P450 catalysts of CPA and IFA 4-hydroxylation are well characterized, the P450 enzyme catalysts of the alternative N-dechloroethylation pathway are poorly defined. Analysis of a panel of fifteen human P450 cDNAs in the baculovirus expression system ('Supersomes') demonstrated that CYP3A4 exhibited the highest N-dechloroethylation activity toward both CPA and IFA, whereas CYP2B6 displayed high N-dechloroethylation activity toward IFA, but not CPA. The contributions of each human P450 to overall liver microsomal N-dechloroethylation were calculated using a recently described relative substrate-activity factor method, and were found to be in excellent agreement with the results of inhibition studies using the CYP3A inhibitor troleandomycin and an inhibitory monoclonal antibody to CYP2B6. With CPA as substrate, CYP3A4 was shown to catalyze >/=95% of liver microsomal N-dechloroethylation, whereas with IFA as substrate, CYP3A4 catalyzed an average of approximately 70% of liver microsomal N-dechloroethylation (range = 40-90%), with the balance of this activity catalyzed by CYP2B6 (range = 10-70%, dependent on the CYP2B6 content of the liver). Because CYP2B6 can make a significant contribution to human liver microsomal IFA N-dechloroethylation, but only a minor contribution to IFA 4-hydroxylation, the selective inhibition of hepatic CYP2B6 activity in individuals with a high hepatic CYP2B6 content may provide a useful approach to minimize the formation of therapeutically inactive but toxic N-dechloroethylated IFA metabolites.

Anti-Bacterial Agents↗

PubMed2000

Growth hormone pulse-activated STAT5 signalling: a unique regulatory mechanism governing sexual dimorphism of liver gene expression.

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release; this release is intermittent in male rats and nearly continuous in females. Comparisons of liver nuclear protein tyrosine phosphorylation in male and female rats have led to the discovery that the liver transcription factor STAT5b is tyrosine phosphorylated in male but not female rats in response to GH pulses. Intermittent plasma GH pulses trigger a rapid and repeated tyrosine phosphorylation and nuclear translocation of liver STAT5b in intact male rats, while the more continuous pattern of GH exposure down-regulates the STAT5b signalling pathway in female rat liver. The central importance of STAT5b for the physiological effects of GH pulses has been verified using a mouse gene knockout model. STAT5b gene disruption leads to a major loss of multiple sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression are decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products are increased in males to near female levels. STAT5b is thus a liver-expressed, latent cytoplasmic transcription factor that undergoes repeated tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation, and is a key intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene transcription. Other studies indicate, however, that STAT5a and STAT5b are both required for constitutive expression in female, but not male liver, of certain GH-regulated CYP enzymes. GH activation of both STAT5 proteins, which in turn form distinct homodimeric and heterodimeric DNA-binding complexes, is thus an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.

Animals↗

PubMed2000

Pulsatility of growth hormone (GH) signalling in liver cells: role of the JAK-STAT5b pathway in GH action.

The intracellular signalling molecule and transcriptional activator STAT5b is a key mediator of the effects of intermittent plasma growth hormone (GH) pulses on the male-specific pattern of liver gene expression and pubertal body growth rates in rodents. Experiments with Stat5b gene-knockout mice have revealed that these GH-regulated, male-specific phenotypes are a direct consequence of GH pulse-dependent STAT5b activation and that loss of function of STAT5b cannot be compensated for by the closely related signalling molecule STAT5a. Physiological plasma GH pulses are required to obtain the high levels of activated STAT5b seen in the livers of males, and down-regulation of the GH receptor (GHR)-JAK-STAT5b pathway in hepatocytes exposed to GH in a near-continuous fashion underlies the low level of liver STAT5b activity that is characteristic of adult female rats. Termination of nuclear STAT5b signalling occurs at the conclusion of a plasma GH pulse, with STAT5b deactivation catalysed by a tyrosine phosphatase. In males, termination of the intracellular signalling stimulated by a plasma GH pulse is proposed to be additionally facilitated by GH-STAT5b-inducible SOCS-CIS proteins, which block the further activation of STAT5b by binding to and inhibiting the action of the GHR-JAK2 complex via multiple mechanisms. In this manner, the liver cell is rendered temporarily unresponsive to further GH-signalling events. SOCS-CIS proteins synthesized in liver cells stimulated continuously with GH may also contribute to the apparent down-regulation of STAT5b signalling that is observed in the female rat liver.

Animals↗