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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.

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.

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.

The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL−/−) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL −/−, undetectable PRL; pituitary grafted PRL−/− (PRL−/−Graft), elevated PRL; and PRL+/−, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL−/− mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL−/−Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL−/− thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL−/− mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL−/−Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL −/−Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.

There have been few studies aimed at determining the effects of maternal peptide hormones on the developing fetus and even fewer aimed at determining the long-term consequences of abnormalities in maternal hormone exposure. In this study, we have examined the effect of maternal prolactin (PRL) on the production, seeding and long-term function of a T lymphocyte subset for which the precursors are only present during fetal life. Using this system, we can determine long-term consequences of maternal hormone exposure without concern for the subsequent influence of the offspring's endocrine milieu. Recombinant versions of the two major forms of the pituitary hormone, PRL, were administered to rats throughout pregnancy. Administration of a molecular mimic of phosphorylated PRL (PP-PRL) resulted in a marked increase in the level of apoptosis in the thymus of newborn pups, an effect that was not duplicated by administration of unmodified PRL. The increased thymic apoptosis in the animals exposed to PP-PRL resulted in decreased epidermal seeding of gammadeltaT cells and a markedly decreased gammadeltaT cell-modulated epidermal response in the offspring. This decreased gammadeltaT cell modulated response persisted to adulthood. We conclude that maternal PRL composition during pregnancy can have a permanent effect on at least one component of the developing immune system.

The importance of prolactin (PRL) in mammopoiesis and milk production is undisputed. However, previous studies investigating the role of PRL in immune function have yielded inconsistencies. These inconsistencies have led to our hypothesis that the immunomodulatory effects of PRL are only manifest under conditions in which the organism is subjected to stress. Thermal injury is a well-known stressor. The goal of this study was to determine whether the lack of PRL enhanced the negative effects of thermal injury-induced immune alterations utilizing a mouse model in which the PRL gene had been disrupted. Mice received either sham or burn treatment, and were sacrificed 4 days later. The immune parameters studied were the capacity of bone marrow cells to form granulocyte-macrophage colony forming units (GM-CFU) in the presence of granulocyte-macrophage colony stimulating factor, and the ability of the splenic T lymphocytes to proliferate in response to phytohemagglutin (PHA). As shown by others, our results reveal that burn increased the number of GM-CFU compared to sham controls; however, this elevation was only significant in the PRL-/- mice. Thermal injury increased PHA-stimulated proliferation of splenic T lymphocytes, however this increase was only significant in the PRL+/- group. We conclude that under conditions of a controlled stress event (thermal injury) [a] the increase in the GM-CFU is exaggerated in the absence of PRL, and [b] the enhancement of PHA-induced proliferation of splenic lymphocytes required PRL. This study supports the hypothesis that the immunomodulatory effects of PRL are manifest when the organism is subjected to stress.

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.

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-meduated expression of pim -1 appears to reflect signaling by MAPK, Jak2, and PI3-kinase.

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.

The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to γ-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2–11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5′-p...

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.

Virtually all cells respond to heat stress by increased expression or induction of one or more of the highly conserved cellular stress response proteins, heat shock proteins (HSPs). Here, we report the unusual property of rat Nb2-11 cells, a prolactin-dependent pre-T-cell line, to display reduced HSP expression following exposure to elevated temperature. After heat stress (41 degrees C, 1 h), there was no evidence of inducible members of the 70 kDa HSP family, a response common to other cell culture and tissue systems. Moreover, expression of constitutive members of the HSP70 and HSP90 families decreased during the heat stress, apparently reflecting a decrease in mRNA stability. Gel shift assays revealed that heat shock factor (HSF) was activated in spite of the lack of expression of inducible HSP70 transcripts, although its DNA binding rapidly deteriorated. Immunoblotting, using an antibody specific to HSF1, indicated that proteolysis of HSF1 may be responsible for this rapid termination of heat shock element binding. CCAAT binding, a component of constitutive HSP70 expression, was also reduced by heat stress in Nb2-11 cells and may account for the decline in constitutive HSP70 expression. Prolactin pretreatment prevented the fragmentation of HSF1, protected heat shock element and CCAAT binding, prevented the decline in constitutive HSP70 and HSP90 expression, and restored a modest expression of inducible HSP70 following heat treatment. Results of this study describe a unique regulatory defect in HSP expression in Nb2-11 cells, possibly a common characteristic of other hormone-dependent tumors.

The breast cancer cell genome is remarkably unstable, most likely due to early dysfunction of DNA replication, repair or recombination (Roskelley and Bissel, 2002). Accumulation of genetic alterations in the cells and/or stroma lead to development of a genetically diverse cell population characterized by uncontrolled cell proliferation (Witz, 2002; Gupta et al., 2006). Therefore, identification of genes that may be responsible for pre-disposition or facilitate progression of the disease may contribute to improvement of currently available therapeutic approaches in treatment of breast cancer. We previously reported the identification, cloning and functional characterization of HRPAP20, which encodes for protein designated Hormone Regulated ProliferationAssociated Protein 20 (accession number: NM_ 014165; Karp et al., 2004). Our observations indicated that HRPAP20 is a regulator of proliferation, survival, and invasion in hormoneresponsive breast cancer cells. Moreover, highly invasive, hormone unresponsive breast cell lines, such as MDA-MB-231 and tumor specimens of invasive breast adenocarcinomas from patients exhibited constitutively elevated levels of HRPAP20 (Karp et al., 2007). Results from an independent study conducted by another group suggested that HRPAP20 is a promising marker of tamoxifen resistance in women with ER alpha-positive breast tumors (Tozlu-Kara et al., 2007). Together, these observations suggested that elevated HRPAP20 may facilitate breast cancer progression toward a more malignant phenotype. Other studies from our group suggested that an interaction between HRPAP20 and calmodulin (CaM) may contribute to HRPAP20-mediated biological effects in tumor cells. Furthermore, a basic amino acid residue (K73) within the putative CaM-binding domain of HRPAP20 appeared to be important for CaM-binding to the protein. CaM has been shown to influence cell cycle control and proliferation in human breast cancer cells by activating CaM-kinase II (CaMK II) and MAPK-mediated signaling pathways (Cheung , 1980; Wang et al., 1983; Rodriguez-Mora et al., 2005). The recent identification of the ErbB2/HER2/Neu, ER-┙, and androgen receptor (AR) as CaM-binding proteins, has opened new areas of investigation in the regulation of signaling by CaM, particularly in hormone-responsive cancer (Cifuentes et al., 2004; Li et al., 2006; Maximciuc et al., 2006). Overexpression or constitutive activation of the epidermal growth factor receptor (EGFR) is frequently associated with the development and progression of a number of human cancers

Previous studies have indicated that prolactin (PRL) interacts with specific, high affinity, immunoreactive binding sites within isolated rat hepatocyte nuclei. Moreover, endogenous PRL appears to be bound to this site. However, it remained important to demonstrate nuclear PRL receptors and hormonal translocation in an intact cell system. Therefore, we sought nuclear translocation of PRL and its receptor in the nucleus of PRL-dependent Nb2 node lymphoma cells. Utilizing immunofluorescence (IF) microscopy, growth-arrested cells were found to constitutively express the PRL receptor in the nucleus and in the membrane/cytosol compartments. Addition of PRL stimulated rapid hormone internalization followed by translocation to the nucleus within 6-12 hrs. The translocation of PRL was found to be reversible and dependent upon ATP. These results indicate that an early event coupled to the mitogenic action of PRL in Nb2 cells is hormone transport to the nucleus during the G1 and S phases of cell cycle. Once in the nucleus, PRL bound to its receptor may directly influence gene transcription.

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.

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 GR alpha and estrogen receptor ER alpha on the transcriptional activation of PXR. The cotransfection of GR alpha in LS174T cells augmented PXR activation by tamoxifen and 4OHT. On the other hand, the presence of ER alpha 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.

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.

Malignant progression of lymphoma cells is associated with acquisition of the cystine/glutamate antiporter, xc-, enhancing cystine uptake. Recently, we showed that sulfasalazine (SASP) is a specific xc- inhibitor. Here, we investigated xc- in mammary cancer cell lines.Expression and function of xc- were evaluated by RT-PCR and 35S-cystine uptake analysis.Xc- expression was elevated 4-fold (p0.001) in cells of the most malignant line, MDA-MB-231, associated with increased 35S-cystine uptake (p0.001). Proliferation was inhibited by 0.2-0.5 mM SASP. 2-Mercaptoethanol (60 microM), a cystine uptake enhancer, completely prevented SASP-mediated growth inhibition in MDA-MB-231 cultures, but only partially in 184A1 and MCF-7 cultures. SASP-induced growth arrest was reversible and not cell cycle-specific.The results suggest: (i) malignant progression of human mammary cancer may be associated with acquisition of xc- expression potentially leading to increased growth autonomy and drug resistance, (ii) xc- may act as a therapeutic target.

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 G(0)-G(1) 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 G(1) 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 (P0.01) increased proliferation in the absence of hormonal stimulation and inhibited apoptosis induced by lactogen deprivation (P0.001). 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 D(3)). 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 (P0.05) increased HRPAP20 during G(1) 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 (P0.006) increased proliferation in the absence of hormone stimulation and augmented survival in the absence of serum (P0.05). We conclude that HRPAP20 is a phosphoprotein that is required for proliferation and survival of hormone-dependent tumor cells.

In this study, we sought to determine if prolactin (PRL) had any influence on burn-induced alterations in myelopoiesis and serum IL-6, IL-10, IL-12, IFN-gamma, TNF-alpha, and MCP-1 levels. To do this, we used mice that were PRL normal, PRL deficient, or hyperprolactinemic and had received a 15% total body surface area burn, sham treatment, or no treatment. We performed clonogenic assays of bone marrow cells, and we found that sham treatment significantly decreased monocyte/macrophage (M) colony formation relative to the control group in the PRL-deficient and PRL-normal mice (P < 0.01). Hyperprolactinemia attenuated the sham-induced decrease in M colony formation. Burn injury significantly increased M colony formation relative to the sham group with an equal significance in the PRL-deficient and PRL-normal mice (P < 0.05). We also showed that burn led to a significant increase in GM colony formation relative to the sham group. This burn-induced increase was significant in the PRL-normal (P < 0.05) and the PRL-deficient (P < 0.01) mice. In the PRL-normal mice, burn injury caused a 2.1-fold increase in the GM colony number, whereas in the PRL-deficient mice burn led to a 2.6-fold increase in GM colony number. When comparing the effects of burn injury on colony formation to the control groups, there were no significant differences seen, irrespective of the PRL level. We observed that all of the cytokines studied, with the exception of IL-10, were influenced by either sham treatment, burn injury, or both forms of stress. This stress-induced response occurred most often in animals that were either hypo- or hyperprolactinemic. We conclude that the PRL level was able to influence the sham-induced and burn-induced alterations in GM and M colony formation. Under euprolactinemic conditions, mice exhibited less often with stress-induced serum cytokine level alterations. We did not find any significant correlations with any of the serum cytokine levels and the ability to form colonies. Importantly, the sham treatment led to immune alterations independent of, and sometimes opposite of burn-induced effects.

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. Methods Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. Results Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation. Conclusions The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.

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.

Previously we showed that the distal element (DE) (−427 to −336 bp) within the pim-1 promoter appeared to regulate its prolactin (PRL)-induced transcription. To determine which specific DE sequences conferred PRL responsiveness, seven 12-bp deletion mutants ligated upstream of the chloramphenicol acetyltransferase gene were transfected into FDC/Nb2 cells. Results from promoter/reporter studies showed that sequential 12-bp deletions of the DE significantly (p

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.

Previously, we reported that activation of the human prolactin receptor (PRLR) produced a protein phosphorylation pattern strikingly similar to that provoked by Concanavalin A (Con A), an activator of the T-cell antigen receptor (TCR). These results suggested that certain signaling components of the TCR may be shared by the activated PRLR. Additional studies here assessed the levels of TCR expression following PRLR stimulation and the effect of TCR activation on PRL-stimulated proliferation in lactogen-dependent pre-T Nb2-11 lymphoma cells. The results indicated that the TCR was expressed on the surface of approx 4% of exponentially proliferating and prolactin- (PRL) treated cells. In contrast, approx 45% of quiescent cells, cultured in the absence of PRL for 24 h, expressed the TCR at the cell surface, suggesting that lactogen withdrawal may up-regulate TCR cell-surface expression. Moreover, TCR activation with anti-CD3 antibodies attenuated PRL-stimulated Nb2-11 cell proliferation in a concentration-dependent manner. In other experiments, immunoprecipitation and immunoblotting of Nb2-11 lysates revealed that activation of the PRLR resulted in rapid tyrosyl phosphorylation of ZAP-70, a critical TCR-associated tyrosine kinase. In addition, ZAP-70 was found to associate transiently with the putative guanine nucleotide exchange factor and substrate, Vav, in PRL-treated cells. ZAP-70 was also found to associate constitutively with the PRLR; PRL stimulation provoked the transient recruitment of Vav to the complex. These observations suggest that PRL signaling reflects the transient formation of a PRLR-ZAP-70-Vav complex and its immunomodulatory actions involve diverse interactions that affect TCR expression and signaling mechanisms.

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.

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.

The Nb2 rat lymphoma represents a useful model for investigation of molecular events coupled to PRL-induced proliferation. Moreover, recent evidence has also demonstrated the utility of this system to study mechanisms linked to programmed cell death (apoptosis). Thus, glucocorticosteroids activate apoptosis in lactogen-dependent Nb2 cells, whereas the addition of PRL abrogates this effect. The present study was conducted to determine whether PRL stimulation in lactogen-dependent Nb2-11 or autonomous Nb2-SFJCD1 cultures alters expression of bcl-2 or bax, genes that suppress or facilitate apoptosis, respectively. We demonstrate that PRL stimulation in stationary Nb2-11 cultures significantly increased the level of bcl-2 messenger RNA (mRNA) within 3 h (15-fold) and its protein product by 6 h, time points previously shown to correspond with G1 cell cycle progression. In Nb2-SFJCD1 cells, bcl-2 mRNA was found to be constitutively present. Addition of PRL to these lactogen-independent cultures further enhanced its expression at the mRNA and protein levels with a kinetic pattern similar to that observed in the PRL-dependent line. Results from stability studies indicated that increased bcl-2 mRNA evoked by PRL in Nb2 cell lines was most likely not attributable to increased stability of its transcripts. Furthermore, the rapid increase in its expression in PRL-stimulated Nb2-11 cells was not altered by inhibition of protein synthesis suggesting a direct action of the hormone. PRL also increased bax mRNA by 8 h in Nb2-11 cultures. However, hormone stimulation markedly attenuated the level of the Bax protein from 2-6 h. In contrast, bax expression in Nb2-SFJCD1 cultures remained unaltered by the addition of the hormone. These results demonstrate that altered expression of bcl-2 and bax are each associated with PRL-stimulated cell cycle progression in Nb2 cells. Moreover, bcl-2 appears to be an immediate-early gene induced by PRL in the hormone-dependent line and may represent an important regulator of early G1 cell cycle progression most likely by suppressing cell death mechanisms.

Previously we demonstrated that lactogen-dependent Nb2 cells express a nuclear prolactin (PRL) receptor. Thus, the nuclear receptor expressed in PRL-dependent Nb2-11 and -independent Nb2-SFJCD1 cells was characterized. Initially, the potential proteolytic processing of internalized 125I-rPRL was investigated. Radiolabeled hormone eluted from a Sephadex G-100 column with a retention time identical to that found for stock hormone, indicating that nuclear PRL was intact. Experiments to investigate the nuclear distribution of the hormone demonstrated that 90% of 125I-rPRL bound to chromatin; the remaining was distributed between "sap-protein" and nucleoplasmic fractions. Chromatin-bound PRL was resistant to high salt and detergent extraction indicating a tight association. Immunoprecipitation and immunoblot analysis revealed the PRL receptor to be 62 kDa in each cell line. Affinity crosslinking experiments and immunoprecipitation demonstrated that 125I-rPRL complexed with a protein(s) of similar M(r) in intact cells. 125I-rPRL binding was saturable and of high affinity (Kds of 180 and 170 pM, for Nb2-11 and Nb2-SFJCD1 lines, respectively). PRL binding was competitively inhibited by ovine and bovine PRLs and hGH, but not by rat GH, and by monoclonal antibodies (McAbs) which recognize the lactogen binding site. These results demonstrate that: (1) 125I-rPRL translocates intact to the Nb2 cell nucleus and tightly associates with chromatin; (2) the chromatin receptor specifically binds 125I-rPRL with high affinity; and (3) the chromatin receptor is essentially identical to its membrane counterpart with respect to mass, binding characteristics, and McAb recognition.

The induction of heat shock proteins (HSPs) by cellular stress and activation of the hypothalamic-pituitary-adrenal axis and sympathetic nervous system by physiological stress are biological responses that aid in the maintenance of cellular and organismal homeostasis, respectively. Based on previous studies, we have hypothesized that HSPs play a functional role in neural and endocrine stress response mechanisms in mammalian organisms. To determine the endocrine and/or neural components regulating stress-induced HSP70 expression in vivo, we have employed the long-acting synthetic propylergoline dopamine agonist CQP 201-403 (CQP). We report the novel observation that CQP mimics the effect of restraint stress to induce HSP70 expression in both adrenal gland and aorta of the rat. The presence of CQP-induced HSP70 mRNA and protein was preceded by the activation of a protein factor capable of binding to the heat shock transcriptional control element. CQP-induced HSP70 expression in the adrenal gland was restricted to the cortex, as previously observed in restraint-stressed animals. However, the distribution of expression among the three cortical layers was distinct. Hypophysectomy virtually eliminated the effects of CQP on the adrenal gland and markedly reduced HSP70 induction in the aorta. Collectively, these results provide evidence that dopaminergic systems contribute to the physiological regulation of HSP70 expression in adrenal gland and aorta directly through actions on receptors in responsive tissues and/or indirectly through the release of pituitary hormones.

Nb2-11 cells, a prolactin (PRL)-dependent T-lymphoma cell line, display an unusual response to heat stress characterized by the lack of expression of inducible hsp70 mRNA transcripts and a reduction in the levels of constitutively expressed heat shock protein (HSP) genes. This aberrant heat shock response appears to result from heat-induced proteolytic fragmentation of heat shock factor (HSF). In this report, we have investigated processes that promote HSF fragmentation and identified characteristics of a protease that may be responsible for this effect. Cycloheximide did not affect HSF fragmentation of heat-shocked Nb2-11 cells suggesting that proteases responsible for this proteolysis are constitutively expressed and become activated by the heat shock conditions. PRL protected Nb2-11 cells from heat-induced fragmentation whereas sodium butyrate (NaBT) rendered a fragmentation-resistant cell line (Nb2-SFJCDl cells) sensitive to HSF proteolysis. Heat-induced HSF fragmentation in Nb2-11 cells was not affected by pretreating cultures with several serine protease inhibitors. However, a dose-dependent decrease in HSF fragmentation was achieved by pretreating cultures with iodoacetamide, a cysteine protease inhibitor that is active in apoptosis. Apparently, the heat shock response in Nb2 cells is attenuated by a mechanism that involves the premature deactivation of HSF by its selective proteolysis. Attenuation of this critical cellular stress response may be an important contributor to the progression of hormone-dependent tumors possibly by influencing apoptotic processes known to regulate the activity of these cells.

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.

1264 CYCLICAL SERUM 25-HYDROXYVITAMIN D PARALLELLING SUNSHINE EXPOSURE IN EXCLUSIVELY BREAST-FED INFANTS: MIRROR IMAGE IN SUMMER VS WINTER BORN