Donna J. Buckley

Induction of CYP3A4 by Efavirenz in Primary Human Hepatocytes: Comparison With Rifampin and Phenobarbital

The antiretroviral agent efavirenz enhances the systemic clearance of coadministered drugs that are cytochrome P450 (CYP) 3A4 substrates. The mechanism of the apparent increase in CYP3A4 activity by efavirenz and the magnitude of change relative to other known inducers are not known. The authors tested the hypothesis that increased enzymatic activity by efavirenz entails CYP3A4 induction and activation of the human pregnane X receptor (hPXR), a key transcriptional regulator of CYP3A4. Employing primary cultures of human hepatocytes, they compared the CYP3A4 inductive effects of efavirenz (1–10 μM) to rifampin (10 μM) and phenobarbital (2 mM). A cell‐based reporter assay was employed to assess hPXR activation. The authors observed that efavirenz caused a concentration‐dependent CYP3A4 induction and hPXR activation. Based on the CYP3A4 activity assay, the average magnitude of induction by efavirenz (5–10 μM) was approximately 3‐to 4‐fold. In comparison, phenobarbital (2 mM) and rifampin (10 μM) caused a 5‐ and 6‐fold induction, respectively.

Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1, bcl-2, and bax expression.

Lactogen-dependent Nb2 lymphoma cells, widely employed for studying prolactin (PRL) mitogenic mechanisms, are also useful for investigations of apoptosis in T-lineage lymphocytes. Utilizing PRL-dependent Nb2-11 cultures, apoptosis-regulatory genes were evaluated for participation in dexamethasone-(DEX) provoked cell death or its inhibition by PRL. Treatment of lactogen-starved, G1-arrested Nb2-11 cells with DEX (100 nM) activated apoptosis within 12 h evaluated by flow cytometric analysis of fragmented DNA. This effect was not associated with altered expression of bcl-2, bax, or pim-1. PRL (10 ng/mL), coincubated with DEX-treated cells, completely blocked DEX-induced apoptosis. This inhibition was associated with increased expression of bcl-2 and pim-1 mRNAs, genes reported to suppress apoptosis, within 2–6 h after addition of the hormone. Moreover, the increased transcription of bcl-2 and pim-1 was coupled to increases in their protein levels. The results suggest that bcl-2, bax, and pim-1 do not play a critical role in DEX-induced apoptosis in Nb2 cells. However, expression of bcl-2, together with pim-1, may have a role in mediating the antiapoptotic actions of PRL.

Dose‐dependent Induction of Cytochrome P450 (CYP) 3A4 and Activation of Pregnane X Receptor by Topiramate

Summary:  Purpose: In clinical studies, topiramate (TPM) was shown to cause a dose‐dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription.

Increased cystine uptake capability associated with malignant progression of Nb2 lymphoma cells

Analysis of rat, pre-T cell ‘Nb2 lymphoma’ sublines, manifesting different degrees of malignant progression, can indicate phenotypic changes potentially useful as therapeutic targets. In this study, the prolactin (cytokine)-dependent Nb2-11 and autonomous Nb2-SFJCD1 sublines were compared for in vitro thiol growth requirements. Whereas Nb2-11 culture growth depended on 2-mercaptoethanol (2-ME; 33–100 μ M), Nb2-SFJCD1 cells were 2-ME-independent. This difference stemmed from differential uptake of exogenous L-cystine, critically required for proliferation. Uptake of 35S-L-cystine (10  μ Ci/ml; 40 μ M) showed Nb2-11 cells had low cystine uptake capability; 2-ME enhanced cystine uptake to growth-sustaining levels. Nb2-SFJCD1 cells did not require 2-ME due to intrinsic, 11-fold higher cystine uptake via the xc- cystine/glutamate transport system. In absence of 2-ME, monosodium glutamate abrogated Nb2-SFJCD1 proliferation by specifically inhibiting cystine uptake (85% at 10 m M). Elevated glutathione (GSH) levels were not essential for growth of either line as shown with L -buthionine-(S,R)-sulfoximine (0.1–4 m M) treatment. The cyst(e)ine requirement therefore did not primarily involve maintenance of normal GSH levels, reported critical for T lymphocyte replication. These and other results suggest increased cystine uptake capability constitutes another potential step in progression of T cell cancers which is not coupled to cytokine autonomy or metastatic ability development. The xc- transport system apparently provides a novel target for T cell cancer therapy. Its inhibition would suppress cystine uptake by certain progressed cells, and also interfere with cystine uptake, and subsequent cysteine release, by eg macrophages, thought to have a role in cysteine delivery to lymphoid cells.

Sulfasalazine-Induced Reduction of Glutathione Levels in Breast Cancer Cells: Enhancement of Growth-Inhibitory Activity of Doxorubicin

Background: We previously showed that the anti-inflammatory drug, sulfasalazine (salicylazosulfapyridine, SASP), can arrest proliferation of MCF-7 and MDA-MB-231 mammary cancer cells by inhibiting uptake of cystine via the xc– cystine/glutamate antiporter. Here we examined SASP with regard to reduction of cellular glutathione (GSH) levels and drug efficacy-enhancing ability. Methods: GSH levels were measured spectrophotometrically. Cellular drug retention was determined with 3H-labeled methotrexate, and drug efficacy with a colony formation assay. Results: Incubation of the mammary cancer cells with SASP (0.3–0.5 mM) led to reduction of their GSH content in a time- and concentration-dependent manner. Similar to MK-571, a multidrug resistance-associated protein inhibitor, SASP increased intracellular accumulation of methotrexate. Preincubation of cells with SASP (0.3 mM) significantly enhanced the potency of the anticancer agent doxorubicin (2.5 nM). Conclusions: SASP-induced reduction of cellular GSH levels can lead to growth arrest of mammary cancer cells and enhancement of anticancer drug efficacy.

Inhibition by genistein of prolactin-induced Nb2 lymphoma cell mitogenesis

Tyrosine kinase activation in mediating the mitogenic action of prolactin (PRL) has been evaluated. Use was made of genistein, a tyrosine kinase antagonist, and cultured rat Nb2 lymphoma cells, i.e. the lactogen-dependent Nb2-11 line and a lactogen-independent subline, Nb2-SFJCD1. Genistein was found to be a potent growth-inhibitor for both lines, inhibiting 3H-thymidine incorporation in Nb2-11 and Nb2-SFJCD1 cells with IC50s of 4.2 and 6.7 micrograms/ml, respectively. Genistein also inhibited expression and translation of the heat shock protein 70 gene and pp40 protein substrate phosphorylation which, in Nb2-11 cells, followed PRL addition within minutes. Genistein inhibition of DNA synthesis in G1-arrested Nb2-11 cells was most pronounced if the agent was added within 1 h of PRL treatment. The results indicate that, while both Nb2 cell lines have a general growth requirement for tyrosyl phosphorylation, the early, PRL-induced tyrosine kinase activation is a component of the PRL mitogenic signal transduction pathway.

Changes in glutathione redox cycling and oxidative stress response in the malignant progression of NB2 lymphoma cells.

Differential analysis of closely related Nb2‐lymphoma cell lines can be used for identification of changes in biochemical properties associated with the malignant progression of certain T‐cell cancers. As tumors progress, they tend to show metabolic alterations such as an increased resistance to oxidative stress, a characteristic that may be correlated with changes in intrinsic antioxidant levels (e.g., glutathione) and in activities of associated enzymes such as the glutathione redox pathway. Whether increases in malignancy of Nb2 cells were associated with changes in cellular glutathione levels and activities of glutathione‐metabolizing enzymes was addressed. To evaluate this relationship, 3 cell lines, showing increased malignancy, were used: Nb2‐U17 (hormone‐dependent, non‐metastatic), Nb2‐11 (hormone‐dependent, metastatic), Nb2‐SFJCD1 (growth factor‐independent, metastatic). Compared to Nb2‐U17 and Nb2‐11 cells, the highly progressed Nb2‐SFJCD1 lymphoma cells maintain low basal glutathione levels. However, the Nb2‐SFJCD1 cells display an enhanced capacity to produce glutathione when challenged with an oxidative stress and show a significantly higher resistance to H2O2‐induced apoptosis. Int. J. Cancer 77:55–63, 1998.© 1998 Wiley‐Liss, Inc.

Prolactin signaling to pim-1 expression: a role for phosphatidylinositol 3-kinase.

Sublines of the lactogen-dependent, rat pre-T Nb2 lymphoma are useful as a model for the investigation of prolactin (PRL) signaling mechanisms, regulation of transcription of target genes, and the immunomodulatory and anti-apoptotic actions of the hormone in T lymphocytes. In the present study, coupling of various tyrosine, serine/threonine, and phospholipid kinase signaling mechanisms to PRL-stimulated Nb2-11 cell proliferation and expression of the protooncogene, pim-1, was investigated utilizing pharmacologic antagonists of a broad spectrum of tyrosine kinases (tyrphostin A25), and the specific enzymes, Jak2 (tyrphostin B42) and ZAP-70 (piceatannol), as well as mitogen-activated protein kinase (MAPK, PD98059), protein kinase C (PKC, calphostin C), and phosphatidylinositol 3-kinase (PI3-kinase, LY294002). Inhibition of each pathway attenuated PRL-stimulated Nb2-11 cell proliferation in a concentration-dependent manner. Blockade of MAPK was the least efficacious; it inhibited proliferation maximally by 60%. Northern blot analysis of pim-1 expression in antagonist-treated cells revealed that MAPK, Jak2 and PI3-kinase appeared to signal to initiation of pim-1 transcription; its expression was attenuated by each of the antagonists. In other experiments, PRL was shown to rapidly activate a downstream effector of PI3-kinase, Akt, and this effect was also blocked by LY294002. It is concluded that PRL-stimulated Nb2 cell proliferation requires participation of each of the signaling pathways investigated. Moreover, hormone-mediated expression of pim-1 appears to reflect signaling by MAPK, Jak2, and PI3-kinase.

Role of Human Pregnane X Receptor in Tamoxifen- and 4-Hydroxytamoxifen-Mediated CYP3A4 Induction in Primary Human Hepatocytes and LS174T Cells

Previously we observed that the antiestrogens tamoxifen and 4-hydroxytamoxifen (4OHT) induce CYP3A4 in primary human hepatocytes and activate human pregnane X receptor (PXR) in cell-based reporter assays. Given the complex cross-talk between nuclear receptors, tissue-specific expression of CYP3A4, and the potential for tamoxifen and 4OHT to interact with a myriad of receptors, this study was undertaken to gain mechanistic insights into the inductive effects of tamoxifen and 4OHT. First, we observed that transfection of the primary cultures of human hepatocytes with PXR-specific small interfering RNA reduced the PXR mRNA expression and the extent of CYP3A4 induction by tamoxifen and 4OHT by 50%. Second, in LS174T colon carcinoma cells, which were observed to have significantly lower PXR expression relative to human hepatocytes, neither tamoxifen nor 4OHT induced CYP3A4. Third, N-desmethyltamoxifen, which did not induce CYP3A4 in human hepatocytes, also did not activate PXR in LS174T cells. We then used cell-based reporter assay to evaluate the effects of other receptors such as glucocorticoid receptor GRα and estrogen receptor ERα on the transcriptional activation of PXR. The cotransfection of GRα in LS174T cells augmented PXR activation by tamoxifen and 4OHT. On the other hand, the presence of ERα inhibited PXR-mediated basal activation of CYP3A4 promoter, possibly via competing for common cofactors such as steroid receptor coactivator 1 and glucocorticoid receptor interacting protein 1. Collectively, our findings suggest that the CYP3A4 induction by tamoxifen and 4OHT is primarily mediated by PXR but the overall stoichiometry of other nuclear receptors and transcription cofactors also contributes to the extent of the inductive effect.

Prolactin Regulation of Apoptosis-Associated Gene Expression in T Cells

Abstract: Evidence accumulated over the last two decades indicates important actions for prolactin (PRL) in regulation of several functions of the immune system. That PRL can serve to facilitate immune cell proliferation is well established. In addition, PRL appears to play a salient role in the genesis and/or potentiation of certain autoimmune diseases. Recent evidence from several laboratories has extended the spectrum of PRL actions in immunological systems to include regulation of lymphocyte pool size through the process of apoptosis. Experimental results obtained using lactogen‐dependent rat pre‐T cell lines, the Nb2 lymphoma, have demonstrated that PRL suppresses cell death mechanisms activated by cytokine/hormone deprivation and cytotoxic drugs such as glucocorticoids. In this paper, we review results from studies conducted to investigate the mechanism(s) underlying PRL‐regulated apoptosis suppression. Effects of the hormone on expression of apoptosis‐associated genes of the Bcl‐2 family as well as the protooncogene pim‐1 in proliferating Nb2 sublines and in cells exposed to apoptotic stimuli are presented. It is concluded that PRL‐mediated apoptosis suppression in immune cells reflects a complex interaction among several gene products.