The human DEK oncogene regulates DNA damage response signaling and repair.

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.

Employing a recombinant HLA-DR3 expression system to dissect major histocompatibility complex II-thyroglobulin peptide dynamism: a genetic, biochemical, and reverse immunological perspective.

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.

Ligand-independent regulation of transforming growth factor beta1 expression and cell cycle progression by the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of its xenobiotic ligands and acts as an environmental checkpoint during the cell cycle. We expressed stably integrated, Tet-Off-regulated AHR variants in fibroblasts from AHR-null mice to further investigate the AHR role in cell cycle regulation. Ahr+/+ fibroblasts proliferated significantly faster than Ahr-/- fibroblasts did, and exposure to a prototypical AHR ligand or deletion of the ligand-binding domain did not change their proliferation rates, indicating that the AHR function in cell cycle was ligand independent. Growth-promoting genes, such as cyclin and cyclin-dependent kinase genes, were significantly down-regulated in Ahr-/- cells, whereas growth-arresting genes, such as the transforming growth factor beta1 (TGF-beta1) gene, extracellular matrix (ECM)-related genes, and cyclin-dependent kinase inhibitor genes, were up-regulated. Ahr-/- fibroblasts secreted significantly more TGF-beta1 into the culture medium than Ahr+/+ fibroblasts did, and Ahr-/- showed increased levels of activated Smad4 and TGF-beta1 mRNA. Inhibition of TGF-beta1 signaling by overexpression of Smad7 reversed the proliferative and gene expression phenotype of Ahr-/- fibroblasts. Changes in TGF-beta1 mRNA accumulation were due to stabilization resulting from decreased activity of TTP, the tristetraprolin RNA-binding protein responsible for mRNA destabilization through AU-rich motifs. These results show that the Ah receptor possesses interconnected intrinsic cellular functions, such as ECM formation, cell cycle control, and TGF-beta1 regulation, that are independent of activation by either exogenous or endogenous ligands and that may play a crucial role during tumorigenesis.

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase.

Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.

Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice.

PURPOSE: To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice. METHODS: Cre driver mice were crossbred with reporter mouse lines (e.g., ZEG and Rosa26R) to obtain Cre/ZEG and Cre/Rosa26R double transgenic mice. The frequency of promiscuous LoxP/Cre recombination was determined by the expression of second reporter genes in the offspring of double transgenic mice. RESULTS: The frequency of promiscuous LoxP/Cre recombination varied in different lines of Cre driver mice and in the sex of the same driver mice with higher penetrance in male than in female double transgenic mice. Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis. Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion. CONCLUSIONS: Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

LS14 cells: a model for chemoresistance in liposarcoma.

Liposarcoma, a malignancy of adipose tissue, is the most common soft tissue sarcoma. Patients whose primary tumor cannot be resected or those who have developed metastasis, have poor prognosis since liposarcomas are highly resistant to chemotherapy. We recently generated a spontaneously immortalized cell line, named LS14, from a patient with metastatic liposarcoma. Our goal was to compare the responsiveness of LS14 and SW872 liposarcoma cells to anti-cancer drugs and explore mechanisms of chemoresistance. Using complementary assays for cell viability and number we found that SW872 cells responded robustly to relatively low concentrations of doxorubicin, cisplatin and vinblastine. This reduction in cell viability was due to apoptosis, as evident by phosphatidylserine exposure and caspase 3 cleavage. In contrast, only a high dose of doxorubicin or combination therapy effectively reduced LS14 cell viability and induced apoptosis. LS14 cells showed a higher expression of Bcl-2 and Bcl-xL, but a lower expression of survivin and Bax, than SW872 cells, suggesting that anti-apoptotic proteins contribute to chemoresistance in LS14 cells. Although LS14 cells did not form colonies in soft agar, they generated large tumors and metastases in SCID mice, establishing their tumorigenicity in vivo. In conclusion, LS14 cells are much more resistant to chemotherapy than SW872 cells, making them an excellent model for exploring the efficacy and mechanism of action of anti-cancer drugs in liposarcomas.

Postburn monocytes are the major producers of TNF-alpha in the heterogeneous splenic macrophage population.

Increased tumor necrosis factor (TNF)-alpha production by postburn splenic macrophages is well documented. Splenic macrophages are a heterogeneous population, and the effect of thermal injury on these subpopulations has not been documented. We examined the effects of scald injury on myeloid cells with the phenotype of red pulp, white pulp, and marginal zone monocyte/macrophages. We found that thermal injury greatly increased the number of splenocytes with the phenotype of white pulp monocytes. These cells were the major producers of TNF-alpha in the postburn spleen. Cells with the red pulp macrophage phenotype had an increased ability to make TNF-alpha after burn injury, but had only half the capacity to make TNF-alpha as did postburn monocytes. The postburn changes in TNF-alpha production correlated with an increased in vivo susceptibility to endotoxin. The increase in monocytes in the spleen from postburn days 1 to 10 correlated with an increasing ability of splenocytes to produce granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and macrophage inflammatory protein 1-alpha. These data suggest that the monocyte is a major source of inflammatory cytokines in the postburn spleen.

Thermal injury elevates the inflammatory monocyte subpopulation in multiple compartments.

Recent publications have demonstrated that human resident and inflammatory monocyte (IM) subpopulations have equivalents in rodents. The effect of thermal injury upon these subpopulations has not been studied. Mice were given a scald burn and killed on postburn days (PBDs) 2, 4, and 8. Bone marrow, blood, and spleen white cells were isolated, and the percentage of resident monocytes (CD11b LY6C), IMs (CD11b LY6C), and monocyte progenitors (macrophage-colony-forming unit [M-CFU]) were determined. The ability of each monocyte population to make TNF-alpha was determined by intracellular cytokine staining. Finally, the ability of sorted fractions from PBD 8 spleen to inhibit lymphocyte proliferation was performed. We noted that there was an increase in M-CFU in the blood and spleen at PBD 8, but the marrow only had a nonsignificant increase in M-CFU. All compartments showed a significant increase in the number of IMs by PBD 8, but no significant changes in resident monocytes were seen. In all compartments, IMs were a major source of TNF-alpha. The postburn increase in IMs and monocyte progenitors in the spleen was accompanied by an increase in the monocyte chemokine monocyte chemoattractant protein 1 and constitutively high levels of the progenitor chemokine stromal-derived factor 1alpha. After burn injury, mice deficient in the receptor for soluble TNF-alpha had equal levels of splenic M-CFU and monocytes, as did wild-type mice, suggesting that this cytokine is not essential for this effect. We conclude that in this model, IMs are a significant source of in vivo TNF-alpha.

Melanoma prevention strategy based on using tetrapeptide alpha-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity.

Melanoma is the deadliest form of skin cancer, with no cure for advanced disease. We propose a strategy for melanoma prevention based on using analogs of alpha-melanocyte stimulating hormone (alpha-MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with alpha-MSH results in stimulation of eumelanin synthesis, reduction of apoptosis that is attributable to reduced hydrogen peroxide generation and enhanced repair of DNA photoproducts. These effects should contribute to genomic stability of human melanocytes, thus preventing their malignant transformation to melanoma. Based on these findings, we synthesized and tested the effects of 3 tetrapeptide alpha-MSH analogs, Ac-His-D-Phe-Arg-Trp-NH2, n-Pentadecanoyl- and 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2, on cultured human melanocytes. The latter two analogs were more potent than the former, or alpha-MSH, in stimulating the activity of tyrosinase, thus melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation (UVR). The above analogs are MC1R agonists, as their effects were abrogated by an analog of agouti signaling protein, the physiological MC1R antagonist, and were absent in melanocytes expressing loss-of-function MC1R. Analogs, such as 4-Phenylbutyryl-His-D-Phe-Arg-Trp-NH2 with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.

Psychogenic stress prior to burn injury has differential effects on bone marrow and cytokine responses.

It is well known that many burn patients experience psychopathological disorders prior to burn injury. However, it is not known whether individuals that have been exposed to chronic psychological stresses will respond differently than unstressed individuals when challenged by a burn injury. In this study, we assessed whether chronic psychogenic stress prior to burn injury had any significant impact on burn injury-induced alterations in the myeloid compartment in the bone marrow and serum cytokine levels utilizing a well-controlled purely psychogenic stress model (predator exposure). Mice were individually caged and exposed to a Long Evans rat for 1 hr a day on 3 consecutive days prior to a 15% total body surface area flame burn. Four days after burn injury, bone marrow and serum were collected to assess myeloid cells and cytokine levels, respectively. Bone marrow cells were cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) to assess clonogenic ability. Flow cytometry was also used to characterize the populations of myeloid cells based on Gr-1 and CD11b staining intensity and to determine the expression of the macrophage colony-stimulating factor receptor (M-CSFR). Serum was assayed for IL-6, IL-12p70, MCP-1, and IFN-gamma by multiplexed sandwich enzyme-linked immunoabsorbent assay (ELISA). We found that predator exposure prior to burn injury ablated the burn-induced increase in myeloid colony formation and attenuated the burn-induced increases in immature monocytes and immature neutrophils in the bone marrow, as well as MCP-1 levels in the serum. Conversely, psychogenic stress exaggerated the burn-induced increase in the number of M-CSFR-positive cells. This study is the first to show the effects of a pure psychogenic stressor (predator exposure) on burn-induced alterations of the immune system. The clinical ramifications of our findings remain to be elucidated.

Stress and prolactin effects on bone marrow myeloid cells, serum chemokine and serum glucocorticoid levels in mice.

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.

Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy.

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.

alpha-Melanocortin and endothelin-1 activate antiapoptotic pathways and reduce DNA damage in human melanocytes.

UV radiation is an important etiologic factor for skin cancer, including melanoma. Constitutive pigmentation and the ability to tan are considered the main photoprotective mechanism against sun-induced carcinogenesis. Pigmentation in the skin is conferred by epidermal melanocytes that synthesize and transfer melanin to keratinocytes. Therefore, insuring the survival and genomic stability of epidermal melanocytes is critical for inhibiting photocarcinogenesis, particularly melanoma, the most deadly form of skin cancer. The paracrine factors alpha-melanocortin and endothelin-1 are critical for the melanogenic response of cultured human melanocytes to UV radiation. We report that alpha-melanocortin and endothelin-1 rescued human melanocytes from UV radiation-induced apoptosis and reduced DNA photoproducts and oxidative stress. The survival effects of alpha-melanocortin and endothelin-1 were mediated by activation of the melanocortin 1 and endothelin receptors, respectively. Treatment of melanocytes with alpha-melanocortin and/or endothelin-1 before exposure to UV radiation activated the inositol triphosphate kinase-Akt pathway and increased the phosphorylation and expression of the microphthalmia-related transcription factor. Treatment with alpha-melanocortin and/or endothelin-1 enhanced the repair of cyclobutane pyrimidine dimers and reduced the levels of hydrogen peroxide induced by UV radiation. These effects are expected to reduce genomic instability and mutagenesis.

Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer.

Prostatic adenocarcinomas depend on androgen for growth and survival. First line treatment of disseminated disease exploits this dependence by specifically targeting androgen receptor function. Clinical evidence has shown that androgen receptor is reactivated in recurrent tumors despite the continuance of androgen deprivation therapy. Several factors have been shown to restore androgen receptor activity under these conditions, including somatic mutation of the androgen receptor ligand-binding domain. We have shown previously that select tumor-derived mutants of the androgen receptor are receptive to activation by bisphenol A (BPA), an endocrine-disrupting compound that is leached from polycarbonate plastics and epoxy resins into the human food supply. Moreover, we have shown that BPA can promote cell cycle progression in cultured prostate cancer cells under conditions of androgen deprivation. Here, we challenged the effect of BPA on the therapeutic response in a xenograft model system of prostate cancer containing the endogenous BPA-responsive AR-T877A mutant protein. We show that after androgen deprivation, BPA enhanced both cellular proliferation rates and tumor growth. These effects were mediated, at least in part, through androgen receptor activity, as prostate-specific antigen levels rose with accelerated kinetics in BPA-exposed animals. Thus, at levels relevant to human exposure, BPA can modulate tumor cell growth and advance biochemical recurrence in tumors expressing the AR-T877A mutation.

Effect of thermal injury on splenic myelopoiesis.

Thermal injury increases the number of macrophage progenitors in the bone marrow but leads to a decrease in the number of granulocyte progenitors. In the spleen, thermal injury increases the numbers of myeloid progenitors, but the lineage commitment of these cells is unknown. In this study mice were given a scald burn, and the number of splenic myeloid progenitors as well as their progeny was determined. BrdU uptake was used to monitor the de novo production of splenocytes for 8 days after the burn. Burn injury increased the numbers of splenic granulocyte-macrophage (GM), granulocyte (G), and macrophage (M) progenitors at postburn day 8 by 12-, 11-, and 18-fold, respectively. Scald injury increased the number of mature PMN (CD11b GR1(bright)) in the spleen and increased the number of white pulp monocyte/macrophages. Increased numbers of BrdU-positive PMN and monocyte/macrophages were seen after injury. Burn macrophages produced increased levels of the anti-inflammatory hematopoietic cytokine G-CSF. Our work clearly shows that the increased myelopoiesis observed postinjury leads to the production of mature myeloid cells. However, the effects of thermal injury on progenitors in the spleen and marrow are not equivalent.

Butylhydroquinone protects cells genetically deficient in glutathione biosynthesis from arsenite-induced apoptosis without significantly changing their prooxidant status.

Arsenic, first among the top environmentally hazardous substances, is associated with skin, lung, liver, kidney, prostate, and bladder cancer. Arsenic is also a cardiovascular and a central nervous system toxicant, and it has genotoxic and immunotoxic effects. Paradoxically, arsenic trioxide is used successfully in the treatment of acute promyelocytic leukemia and multiple myeloma. Arsenic induces oxidative stress, and its toxicity is decreased by free thiols and increased by glutathione depletion. To further characterize the role of glutathione and oxidative stress in the toxicity of arsenic, we have used fetal fibroblasts from Gclm(-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis. Gclm(-/-) mouse embryo fibroblasts (MEFs) are eight times more sensitive to arsenite-induced apoptotic death. Because of a dramatic decrease in glutathione levels, Gclm(-/-) MEFs have a high prooxidant status that is not significantly relieved by treatment with the phenolic antioxidant tBHQ; however, tBHQ blocks arsenite-induced apoptosis in both Gclm(+/+) and Gclm(-/-) cells, although it raises a significant antioxidant response only in Gclm(+/+) cells. Global gene expression profiles indicate that tBHQ is significantly effective in reversing arsenite-induced gene deregulation in Gclm(+/+) but not in Gclm(-/-) MEFs. This effect of tBHQ is evident in the expression of metalloproteases and chaperones, and in the expression of genes involved in DNA damage and repair, protein biosynthesis, cell growth and maintenance, apoptosis, and cell cycle regulation. These results suggest that regulation of glutathione levels by GCLM determines the sensitivity to arsenic-induced apoptosis by setting the overall ability of the cells to mount an effective antioxidant response.

Effects of prolactin level on burn-induced aberrations in myelopoiesis.

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.

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation.

Activation of the melanocortin 1 receptor (MC1R) by α-melanocortin (α-MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)-induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α-MSH. α-MSH up-regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV-induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3-related (ATR) and ataxia telangiectasia mutated (ATM) and their respect-ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild-type p53-induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α-MSH increases UV-induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.

Oral tungstate (Na2WO4) exposure reduces adaptive immune responses in mice after challenge.

Tungstate (WO²â»4) has been identified as a ground water contaminant at military firing ranges and can be absorbed by ingestion. In this study, C57BL6 mice were exposed to sodium tungstate (Na2WO4·2H2O) (0, 2, 62.5, 125, and 200 mg/kg/day) in their drinking water for an initial 28-day screen and in a one-generation (one-gen) model. Twenty-four hours prior to euthanasia, mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 µg/mouse) or saline as controls. After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. In the 28-day and one-gen exposure, statistically significant reductions were observed in the quantities of activated cytotoxic T-cells (TCTL; CD3(+)CD8(+)CD71(+)) and helper T-cells (TH; CD3(+)CD4(+)CD71(+)) from spleens of SEB-treated mice. In the 28-day exposures, CD71(+) TCTL cells were 12.87 ± 2.05% (SE) in the 0 tungstate (control) group compared to 4.44 ± 1.42% in the 200 mg/kg/day (p < 0.001) group. TH cells were 4.85 ± 1.23% in controls and 2.76 ± 0.51% in the 200 mg/kg/day (p < 0.003) group. In the one-gen exposures, TCTL cells were 7.98 ± 0.49% and 6.33 ± 0.49% for P and F1 mice after 0 mg/kg/day tungstate vs 1.58 ± 0.23% and 2.52 ± 0.25% after 200 mg/kg/day of tungstate (p < 0.001). Similarly, TH cells were reduced to 6.21 ± 0.39% and 7.20 ± 0.76%, respectively, for the 0 mg/kg/day P and F1 mice, and 2.28 ± 0.41% and 2.85 ± 0.53%, respectively, for the 200 mg/kg/day tungstate P and F1 groups (p < 0.001). In delayed-type hypersensitivity Type IV experiments, tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day. These data indicate that exposure to tungstate can result in immune suppression that may, in turn, reduce host defense against pathogens.