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Objective: Current evidence supports the conclusion that prolactin (PRL) is not an obligate immunoregulatory hormone and influences the immune system predominantly during stress conditions. In this study, we examined the impact of PRL on the psychogenic stress-induced responses of myeloid cells. Methods: Seven-week-old PRL+/– (normal) and PRL–/– (deficient) mice were exposed to a predator for 1 h/day on 3 consecutive days. Another group of PRL-deficient mice received either 1 pituitary graft (hyperprolactinemic) or sham surgery at 5 weeks of age, while PRL-normal mice only received sham surgery. Two weeks later, these mice were also subjected to predator exposure. One day after the last predator exposure session, all mice were killed and the bone marrow and blood harvested. Results: Significant differences in the myeloid cells between PRL-normal and PRL-deficient mice only occurred in stressed conditions. The median serum corticosterone levels were consistently higher in PRL-deficient mice. The implantation of a pituitary graft lowered the corticosterone levels to those observed in PRL-normal mice. The absolute number of immature neutrophils as well as the numbers of granulocyte macrophage, monocyte/macrophage and granulocyte colonies were significantly higher in the stressed PRL-deficient mice; however, only the increased number of immature neutrophils was reversed by pituitary grafting. Conclusions: Our findings support previous observations that PRL influences myeloid cells of the bone marrow most profoundly in stressed conditions. However, the mechanism by which PRL influences bone marrow myeloid cells during stress cannot be explained solely by its effect on serum corticosterone.

Patients with refractory inflammatory bowel disease (IBD) exhibit increased expression of intestinal P-glycoprotein (P-gp) as well as elevated luminal IFN-γ and nitric oxide (NO) levels. Using the in vitro Caco-2 cell culture model, we investigated whether these pathological mediators associated with the etiology of IBD affect functional activity of intestinal efflux systems. IFN-γ reduced cellular uptake of cyclosporin A (CysA) but not methotrexate (MTX) in a time- and concentration-dependent manner. Simultaneously, P-gp expression increased by approximately twofold. Coincubation with the inducible NO synthase inhibitor l- N6-(1-iminoethyl)lysine (l-NIL) dramatically reduced production of intracellular NO in response to IFN-γ stimulus. The presence of l-NIL also abrogated the cytokine-mediated increase in P-gp expression and function suggesting that NO is required for IFN-γ-mediated activation of this efflux system. Exposure of Caco-2 cells to the chemical NO donor S-nitroso- N-acetylpenicillamine (SNAP) produced a concentration-dependent decrease in intracellular CysA accumulation that was paralleled by an increase in P-gp expression. Both IFN-γ and SNAP enhanced DNA binding of NF-κB, whereas inclusion of l-NIL dramatically decreased this cytokine-induced effect on NF-κB binding. These results suggest that NO mediates IFN-γ-induced increase in expression and function of intestinal P-gp in the human Caco-2 cell culture model by altering DNA binding of NF-κB, which may enhance transcription of the ABCB1 gene encoding for this efflux system.

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 microM) to rifampin (10 microM) 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 microM) was approximately 3- to 4-fold. In comparison, phenobarbital (2 mM) and rifampin (10 microM) caused a 5- and 6-fold induction, respectively.

The prolactin (PRL)-dependent rat Nb2 T lymphoma is a valuable model for investigation of molecular mechanisms that underlie tumor progression in hormone-dependent cancers. mRNA differential display was used to screen for novel gene products expressed in hormone-stimulated or differentiating agent-treated Nb2 sublines. From numerous transcripts identified, DNA sequencing and GenBank analysis revealed a novel 289-bp fragment. Using 5′-rapid amplification of complementary ends-PCR, this fragment was used to clone a unique 2117-bp cDNA, designated HRPAP20 (hormone-regulated proliferation-associated protein), in rat lymphoma cells. Computer-assisted sequence analysis revealed a single open reading frame that encoded a putative 20.2-kDa protein. The effect of hormone stimulation to alter expression of HRPAP20 was evaluated by Northern blot analysis of total RNA obtained from PRL-stimulated, lactogen-dependent Nb2-11 cells. Quiescent cells, synchronized in the G0-G1 phase of cell cycle, exhibited reduced HRPAP20 expression compared with exponentially proliferating cultures. The addition of mitogenic concentrations of PRL to stationary cells increased HRPAP20 mRNA accumulation within 4–6 h, corresponding to G1 cell cycle progression. Immunoblot analysis showed that PRL also increased HRPAP20 protein levels within 4 h. In addition, PRL stimulated serine phosphorylation of the HRPAP20 protein with a similar kinetic pattern. Stable transfection of the HRPAP20 cDNA into Nb2-11 cells significantly (P < 0.01) increased proliferation in the absence of hormonal stimulation and inhibited apoptosis induced by lactogen deprivation (P < 0.001). In the hormone-independent and highly malignant Nb2-SFJCD1 subline, the constitutive expression of HRPAP20 was markedly reduced by exposure of the cells to dietary differentiating agents (butyrate, retinoic acid, and vitamin D3). After removal of these substances, PRL stimulated its expression in a manner similar to that observed in PRL-dependent Nb2-11 cells. HRPAP20 expression was also evaluated in MCF-7 cells. Its expression was detectable in quiescent cultures; addition of PRL significantly (P < 0.05) increased HRPAP20 during G1 cell cycle progression. Exposure of the cells to butyrate or retinoic acid reduced HRPAP20 expression, similar to the effects of these substances in the malignant rat lymphoma. Stable transfection of HRPAP20 into MCF-7 cells significantly (P < 0.006) increased proliferation in the absence of hormone stimulation and augmented survival in the absence of serum (P < 0.05). We conclude that HRPAP20 is a phosphoprotein that is required for proliferation and survival of hormone-dependent tumor cells.

Recent reports have documented a functional deficit of organic cation transport in diabetic rats by an unknown mechanism. This study was designed to test the hypothesis that experimental diabetes decreases expression of organic cation transporters at the basolateral membrane. Streptozotocin-induced diabetic rats were maintained for varying durations after induction of diabetes. A second group of age-matched control rats was maintained in a parallel manner. Kinetic analysis of tetraethylammonium accumulation in freshly isolated proximal tubular cells indicated a significantly lower V(max) value for the diabetics versus controls with no statistical difference in K(m) values between the two groups. Cortex sections were processed by standard procedures for Northern and immunoblot analysis. Protein expression of the organic cation transporters rOCT1 and rOCT2 progressively decreased with increasing duration of diabetes. After 21 days of diabetes, rOCT1 and rOCT2 were maximally reduced by 50 and 70%, respectively. Quantification of mRNA expression revealed that the roct1 transcript remained unchanged, whereas the roct2 transcript was decreased by 50% after 14 days of diabetes. Treatment with insulin prevented the reductions in transporter levels. These results support the hypothesis by demonstrating that experimental diabetes decreased expression of both rOCT1 and rOCT2 protein and also of roct2 mRNA accumulation. On the other hand, roct1 mRNA levels were unaffected by the diabetic state. This suggests that differences in rOCT2 protein may result from transcriptional and/or translational changes, whereas rOCT1 deficits may be due to posttranscriptional alterations.

Tamoxifen is a widely utilized antiestrogen in the treatment and chemoprevention of breast cancer. Clinical studies document that tamoxifen administration markedly enhances the systemic elimination of other drugs. Additionally, tamoxifen enhances its own clearance following repeated dosing. The mechanisms that underlie these clinically important events remain unresolved. Here, we report that tamoxifen and its metabolite 4-hydroxytamoxifen markedly induce cytochrome P450 3A4, a drug-metabolizing enzyme of central importance, in primary cultures of human hepatocytes. Tamoxifen and 4-hydroxytamoxifen (1-10 microM) significantly increased the CYP3A4 expression and activity (measured as the rate of testosterone 6beta-hydroxylation). Maximal induction was achieved at the 5 microM level. At this level, tamoxifen and 4-hydroxytamoxifen caused a 1.5- to 3.3-fold (mean, 2.1-fold) and 3.4- to 17-fold (mean, 7.5-fold) increase in the CYP3A4 activity, respectively. In comparison, rifampicin treatment resulted in a 6- to 16-fold (mean, 10.5-fold) increase. We also observed corresponding increase in the CYP3A4 immunoreactive protein and mRNA levels. Furthermore, tamoxifen and 4-hydroxytamoxifen efficaciously activated the human pregnane X receptor (hPXR; also known as the steroid xenobiotic receptor), a key regulator of CYP3A4 expression. The efficacy of tamoxifen and 4-hydroxytamoxifen relative to rifampicin for hPXR activation was approximately 30 and 60%, respectively. Our results indicate that the mechanism of tamoxifen-mediated alteration in drug clearance pathways in humans may involve CYP3A4 induction by the parent drug and/or its metabolite. Furthermore, the CYP3A4 induction may be a result of hPXR activation. These findings have important implications for optimizing the use of tamoxifen and in the development of newer antiestrogens.

The rat Nb2 lymphoma is useful for studying prolactin (PRL) receptor signaling to mitogenesis and apoptosis suppression. Previous results showed that PRL rapidly induced expression of several apoptosis suppressor genes during the G1 phase of cell cycle in this model. The X-linked inhibitor of apoptosis protein (XIAP) gene product acts to suppress apoptosis by direct inhibition of caspases. The present study was conducted to determine whether PRL alters XIAP expression in lactogen-dependent Nb2-11 or -independent Nb2-SFJCD1 cells. Stationary Nb2-11 cultures expressed detectable levels of an 8.9-kb XIAP transcript. PRL (20 ng/mL) stimulated its expression, reaching maximal levels within 12 h. Expression of XIAP was also evaluated in Nb2-SFJCD1 cells subsequent to treatment with differentiating agents (sodium butyrate [2 mM, 72 h], all trans-retinoic acid [10 microM, 72 h], or 1,25-dihydroxycholecalciferol [100 nM, 24 h]). PRL significantly increased XIAP expression in cells previously treated with these compounds. Further analysis revealed that PRL stimulated XIAP expression during S phase of the cell cycle. To determine whether XIAP suppressed apoptosis, its cDNA was stably transfected into Nb2-11 cells. Compared to controls, cells overexpressing XIAP exhibited substantially less DNA fragmentation when apoptosis was induced by PRL deprivation or glucocorticoids. We conclude that PRL-stimulated XIAP expression likely serves to suppress apoptosis as cells progress through the later phases of the cell cycle.

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.

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 microM), 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 microCi/ml; 40 microM) 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 x(c)- cystine/glutamate transport system. In absence of 2-ME, monosodium glutamate abrogated Nb2-SFJCD1 proliferation by specifically inhibiting cystine uptake (85% at 10 mM). Elevated glutathione (GSH) levels were not essential for growth of either line as shown with L-buthionine-(S,R)-sulfoximine (0.1-4 mM) 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 x(c)- 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.

The lactogen-dependent Nb2 lymphoma line (Nb2-11) represents a useful pre-T cell model for investigation of early molecular events coupled to PRL-stimulated cell cycle progression. Expression of pim-1, a protooncogene that encodes a conserved cytosolic serine/threonine protein kinase, is rapidly induced in hematopoietic cells upon mitogen stimulation and is thought to be important for lymphocyte activation. The present study was conducted to determine whether mitogen stimulation in Nb2-11 or lactogen-independent Nb2-SFJCD1 cells provokes pim-1 gene expression. The pim-1 transcript was undetectable in control growth-arrested Nb2-11 cultures; however, PRL rapidly stimulated its expression in a biphasic manner. Peak expression occurred within 2-4 h (40-fold) and was followed by a second elevation at 12 h. The effect of PRL and IL-2 to induce pim-1 at 2 h was concentration dependent and not inhibited by cycloheximide. In Nb2-SFJCD1 cells, pim-1 messenger RNA was expressed in control cultures and augmented by PRL stimulation. Results from stability studies indicated that the t1/2 values for the pim-1 transcript were 79 and 81 min in PRL-stimulated Nb2-11 cells at 2 and 12 h. However, in the lactogen-treated Nb2-SFJCD1 line, it was nearly 3-fold more stable (219 min) at 2 h compared to that determined at either 12 h or in unstimulated cultures. In other experiments, PRL-stimulated expression of the pim-1 protein was evaluated in [35S]methionine-labeled cells by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Nb2-11 cells, enhanced [35S]pim-1 expression paralleled its messenger RNA transcription through 8 h. Elevated [35S]pim-1 was detected within 1 h and peaked by 2-4 h. Therefore, pim-1 represents an immediate early gene induced by PRL stimulation in Nb2-11 cells. Its initial peak of transcription occurs early during G1 cell cycle progression, whereas a second elevation is coincident with the G1/S transition. These results demonstrate that mitogen-induced expression of pim-1 is a rapid event in Nb2 lymphoma cells and suggest that it may be associated with cell cycle progression.

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 μg/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.

The trophic effects of prolactin (PRL) in rat liver have been linked to activation of protein kinase C (PKC). Since alterations in PKC activity imply its activation by 1,2-diacylglycerol (DAG), we tested whether PRL treatment stimulated DAG generation coupled to induction of a growth response in primary hepatocytes. Addition of PRL to hepatocyte cultures significantly increased [3H]-glycerol incorporation into DAG within 5 minutes which was followed by a loss of cytosolic PKC activity by 10 minutes. Prolactin also significantly enhanced radiolabel incorporation into triacylglycerol and phospholipids within 10 minutes and induced ODC activity at 6 hours. Therefore, prolactin-stimulated alterations in PKC activity are preceded by enhanced DAG generation. Moreover, these events appear to be coupled to PRL-stimulated entry of hepatocytes into cell cycle.

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.

The induction of cytochrome P450 (CYP) 3A4 by drugs and other xenobiotics is a common cause of serious drug interactions. The aim of this study was to comparatively examine the effects of paclitaxel and docetaxel, two structurally related taxane anticancer agents, on the activity and expression of hepatic CYP3A4.Employing primary cultures of human hepatocytes from multiple donors, we investigated the differences in the magnitude of CYP3A4 induction and relative accumulation of paclitaxel and docetaxel. The CYP3A4 activity of intact hepatocytes was measured as the rate of testosterone 6beta-hydroxylation. The CYP3A4-specific immunoreactive protein and mRNA levels were measured employing Western blot and Northern blot analysis, respectively. Furthermore, employing cell-based reporter gene assay in CV-1 cells, we evaluated the capacity of paclitaxel and docetaxel to activate human pregnane X receptor (hPXR), an orphan nuclear receptor that plays a key role in the transcriptional regulation of CYP3A4.In concurrence with previous reports, we observed that paclitaxel potently induced CYP3A4 activity and expression in hepatocytes treated for 48-96 h. However, docetaxel did not increase the activity or the CYP3A4 immunoreactive protein levels for treatment periods up to 96 h. A marginal increase in the CYP3A4 mRNA levels was observed in cells treated with higher levels (5 and 10 microM) of docetaxel. Furthermore, while paclitaxel effectively activated hPXR (the half-maximal effective concentration, EC50, being about 5.2 microM), docetaxel weakly activated hPXR, and moreover the activation occurred only at high concentrations relative to paclitaxel. A comparison of the cellular concentrations of paclitaxel and docetaxel, in the cell culture models employed for evaluating CYP3A4 induction and hPXR activation, revealed that the intracellular paclitaxel levels were three-fold higher than that of docetaxel. Thus, it appears that both pharmacokinetic (drug concentration) and pharmacodynamic differences (hPXR activation) may account for the observed differences in CYP3A induction by paclitaxel and docetaxel.Our studies suggest that docetaxel has markedly reduced propensity to cause drug interactions that may entail hepatic CYP3A4 induction.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.

The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT‐1 in Caco‐2 cells. Uptake kinetics of Gly‐Sar into Caco‐2 cells that were maintained in iso‐osmotic media containing 25 or 5.5mM glucose were determined in the presence and absence of amino acid‐selective chemical modifiers and dithiothreitol. Inhibition of Gly‐Sar uptake into Caco‐2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT‐1. The effect of extracellular glucose on PepT‐1gene expression was assessed using comparative RT‐PCR. Long‐term exposure of Caco‐2 cells to 25mM glucose reduced maximum transport capacity for Gly‐Sar uptake without altering PepT‐1gene expression. In contrast, binding affinity of Gly‐Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V max , while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco‐2 cells with dithiothreitol restored PepT‐1 activity in cells maintained at 25mM glucose. In conclusion, cell culture media containing 25mM glucose decreases maximum transport capacity of PepT‐1 in Caco‐2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.

Analysis of cultured rat "Nb2 lymphoma" cell lines, showing different degrees of malignant progression, can lead to identification of phenotypic changes associated with this phenomenon in T-cell cancers. In the present study we have compared the metastatic sublines, Nb2-11 and Nb2-SFJCD1, with regard to ascorbate and glutathione recycling, important processes in cellular protection from oxidative stresses. Whereas the Nb2-11 subline is prolactin (PRL)-dependent, the genetically related Nb2-SFJCD1 subline is growth factor-independent and shows more chromosomal alterations, indicative of more advanced progression. The Nb2-SFJCD1 cells, compared to the Nb2-11 cells, were less sensitive to toxic effects of dehydroascorbate, a potentially toxic oxidation product of ascorbate. Results were consistent with a significantly higher production of reducing equivalents (e.g., NADPH, GSH) and an accelerated reduction of dehydroascorbate by homogenates of Nb2-SFJCD1 cells. However, the increased resistance was apparently not directly related to the cellular uptake and reduction of dehydroascorbate by whole cells, which was similar in both cell lines. Observations indicate that Nb2 lymphoma cells, in their progression to malignancy, can acquire an enhanced capability to protect themselves from oxidative damage assisting them in withstanding the oxidative stress that anti-neoplastic drugs can cause. The adaptation may also be a mechanism that is utilized by tumor cells in suppressing apoptosis and other protective cellular functions facilitating, or potentiating, a tumor cell's ability to become more metastatic. However, the mechanism leading to this augmented capacity of Nb2 lymphoma cells to resist oxidative stress in not known and is the subject for further study.

The parental rat Nb2 lymphoma is a prolactin (PRL)-dependent T cell line. Exposure of a PRL-independent subline, Nb2-SFJCD1, to sodium butyrate (NaBT) causes transient reversal of their growth factor-independent proliferation in association with constitutive expression of protooncogenes pim-1and c-myc. In the present study, we investigated the effect of NaBT treatment on the sensitivity of Nb2-SFJCD1 cells to dexamethasone (DEX)-induced apoptosis. Pretreatment with NaBT (2 mM, 72 h) partially reversed resistance to apoptosis in Nb2-SFJCD1 cells exposed to DEX (100 nM) for 12 h, assessed by flow cytometric analyses of DNA fragmentation. However, the cytolytic effect of DEX was abrogated by PRL i n a time- and concentration-dependent manner. Eval uati on of apoptosis-associated gene expression in NaBT-pre-treated cultures incubated with DEX or DEX+PRL indicated that the apoptosis resistance did not stem from altered bcl-2 or bax expression. However, there was a strong correlation between the resistance to DEX-activated apoptosis and their enhanced expression of pim-1 mRNA and protein. The results show that it is possible to reverse DEX-induced apoptosis of Nb2 pre-T cells and suggest the pim-1 gene product has an important role as a suppressor of this process, perhaps functioning as a mediator of PRL action.

Chemically induced murine diabetes is associated with negative calcium (Ca) balance & decreased serum 1,25-dihydroxyvitamin D (1,25(OH)2D) levels; insulin therapy corrects these defects. We hypothesized that diabetes in pregnancy causes decreased maternal ionized Ca (Cai) & 1,25(OH)2D, with indirect effects on neonatal Ca, & that prospective strict management of diabetes from early pregnancy ameliorates these changes. In a comprehensive clinical trial, 57 pregnant insulin-dependent diabetics were randomly assigned during the first trimester to strict vs customary management (Sm vs. Cm) grps. Maternal Cai at delivery was higher in Sm (4.52 ± 0.08 SEM mg/dl) vs. Cm (4.27 ± 0.07 mg/dl, p< 0.02). There was no difference between grps. in maternal 25-hydroxyvit. D (25OHD) or 1,25(OH)2D at delivery. Infants (IDMs) in Sm grp. had higher 24 hr. serum Ca (8.3 ± 0.20 mg/dl) vs. Cm grp IDMs (7.8 ± 0.23 mg/dl, p < 0.04), but no difference was noted by 72 hr. There was no difference in Sm vs. Cm neonatal 25OHD (27 ± 2.9 ng/ml vs. 27 ± 3.1) or 1,25(OH)2D (60 ± 9.4 pg/ml vs. 60 ± 6.9) at 24 hrs. Hypocalcemic (Ca < 7.0 mg/dl)2(HC) IDMs had lower maternal Cai (4.15 ± 0.09 mg/dl) vs. nomocalcemic (NC) IDMs (4.46 ± 0.06, p < 0.02); there was no difference in maternal or neonatal 25OHD & 1,25(OH)2 for HC vs. NC infants. Thus, strict management of maternal diabetes results in increased maternal Cai, but has no effect on maternal 1,25(OH)2. We speculate that improved Ca status in the diabetic pregnancy contributes to improved neonatal Ca homeostasis.