The Belgrade (b) rat has an autosomal recessively inherited, microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake and gastrointestinal iron absorption. The b reticulocyte defect appears to be failure of iron transport out of endosomes within the transferrin cycle. Aspects of this phenotype are similar to those reported for the microcytic anemia (mk) mutation in the mouse. Recently, mk has been attributed to a missense mutation in the gene encoding the putative iron transporter protein Nramp2. To investigate the possibility that Nramp2 was also mutated in the b rat, we established linkage of the phenotype to the centromeric portion of rat chromosome 7. This region exhibits synteny to the chromosomal location of Nramp2 in the mouse. A polymorphism within the rat Nramp2 gene cosegregated with the b phenotype. A glycine-to-arginine missense mutation (G185R) was present in the b Nramp2 gene, but not in the normal allele. Strikingly, this amino acid alteration is the same as that seen in the mk mouse. Functional studies of the protein encoded by the b allele of rat Nramp2 demonstrated that the mutation disrupted iron transport. These results confirm the hypothesis that Nramp2 is the protein defective in the Belgrade rat and raise the possibility that the phenotype shared by mk and b animals is unique to the G185R mutation. Furthermore, the phenotypic characteristics of these animals indicate that Nramp2 is essential both for normal intestinal iron absorption and for transport of iron out of the transferrin cycle endosome.

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-γ. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

Purpose Accurate identification of tobacco use is critical to implement evidence-based cessation treatments in cancer patients. The purpose of this study is to evaluate the accuracy of self-reported tobacco use in newly diagnosed cancer patients. Methods Tobacco use questionnaires and blood samples were collected from 233 newly diagnosed cancer patients (77 lung, 77 breast, and 79 prostate cancer). Blood was analyzed for cotinine levels using a commercially available enzyme-linked immunosorbent assay. Patients with cotinine measurements exceeding 10 ng/mL were categorized as current smokers. Smoking status based upon cotinine levels was contrasted with self-report in current smokers, recent quitters (1 or less year since quit), non-recent quitters (>1 year since quit), and never smokers. Multivariate analyses were used to identify potential predictors of discordance between self-reported and biochemically confirmed smoking. Results Cotinine confirmed 100 % accuracy in self-reporting of current and never smokers. Discordance in cotinine and smoking status was observed in 26 patients (15.0 %) reporting former tobacco use. Discordance in self-reported smoking was 12 times higher in recent (35.4 %) as compared with non-recent quitters (2.8 %). Combining disease site, pack-year history, and employment status predicted misrepresentation of tobacco use in 82.4 % of recent quitters. Conclusions Self-reported tobacco use may not accurately assess smoking status in newly diagnosed cancer patients. Patients who claim to have recently stopped smoking within the year prior to a cancer diagnosis and lung cancer patients may have a higher propensity to misrepresent tobacco use and may benefit from biochemical confirmation.

No abstract

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was approximately 20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1.

Tobacco use significantly increases the risk of developing cancer. Moreover, there is growing evidence that tobacco use decreases survival in cancer patients. Nicotine, a systemically available component of tobacco, is associated with tumor promotion and decreased apoptosis in cell culture; however, the role of nicotine on response to radiotherapy (RT) or chemoradiotherapy (CRT) in vivo has not been evaluated. Our study evaluated the effects of nicotine administration on cancer cell survival in cell culture and mouse models. Nicotine increased survival in two cell lines following RT in vitro. Nicotine administration in mice during fractionated RT or CRT increased xenograft regrowth as compared to RT or CRT alone. Nicotine increased hypoxia-inducible factor 1-alpha (HIF-1a) expression in tumor xenografts without altering expression of carbonicanhydrase, a clinical marker of tumor hypoxia. The effects of nicotine on HIF-1a expression were transient, returning to baseline levels within 2-3 days after nicotine removal. Further mechanistic studies indicated that inhibition of phosphoinositide-3-kinase (PI3K) prevented nicotine-mediated increases in HIF-1a expression as well as the prosurvival effects of nicotine on RT. These findings imply that during tobacco use, nicotine may function as a systemic agent through acute and reversible regulation of HIF-1a expression and a decreased therapeutic response.Tobacco use is the most significant preventable risk factor for the development of lung cancer and continued tobacco use during cancer treatment is associated with poor therapeutic outcomes. 1-3 Analysis of the effects of tobacco use in cancer patients demonstrates that smoking decreases survival in both tobacco-related and nontobacco-related cancers. 4 Importantly, the effects of tobacco use appear to be systemic rather than localized and thus supporting the role of a systemically available agent as a mediator of the adverse effects of tobacco.Nicotine is a systemically available agent of tobacco that is currently advocated as a clinical standard of care for smoking cessation. 5 As one of more than 7,000 compounds in tobacco smoke, nicotine has the capacity to penetrate all tissues in the body and bind to a spectrum of nicotinic acetylcholine receptors (nAChR) present on both normal (noncancerous) and cancerous tissue. Nicotinic activation of nAChRs in cancerous tissue leads to broad downstream activation of several tumor promoting proteins including activation of the phosphoinositide kinase-3 (PI3K)-Akt cascade as well as the Ras-Raf-MEK-ERK1/2 cascade, resulting in increased proliferation, angiogenesis, invasion, metastasis and decreased apoptosis. [6][7][8] In parallel, nicotine administration has been shown to decrease the cytotoxic effects of chemotherapy and radiotherapy (RT) in vitro 9-12 ; however, no studies have evaluated the effects of nicotine on therapeutic response in vivo.Separate studies demonstrate that nicotine administration significantly increases hypoxia inducible factor-1 alpha (HIF1a) in vitro. 1...

Calcitriol (1,25-dihydroxycholecalciferol) has antiproliferative and/or proapoptotic effects on many cell types and the glucocorticoid dexamethasone enhances these effects. We have shown that calcitriol modulates several key signaling proteins involved in differentiation, proliferation and apoptosis in tumor-derived murine endothelial cells (TDEC) and that these effects were not seen with endothelial cells isolated similarly from normal tissues. In the present study, TDEC and mouse embryonic yolk sac endothelial cells (MYSEC) were treated with calcitriol and followed over time for an effect. MYSEC were utilized as ‘normal’ control endothelial cells because they were more primitive, being isolated from a highly neovascular tissue, and had a similar morphology without the stimulus of the tumor microenvironment. The vitamin D receptor (VDR) is present in TDEC and MYSEC, and was upregulated in calcitriol-treated TDEC and MYSEC; dexamethasone further increased VDR expression following 48 h of treatment. The modulatory effects on signaling proteins were maximal by treatment for 48 h; phospho-Erk, phospho-Akt, p21 and bcl-2 were decreased in treated TDEC with the induction of p27 but there were no effects on MYSEC. After 48 h increased apoptosis was seen in treated TDEC by annexin V labeling with caspase-3 cleavage and decreased levels of poly(ADP-ribose) polymerase, but no effects were seen in MYSEC. Cell cycle analysis showed increased G₀/G₁ arrest and an increase in the apoptotic sub-G₁ peak in treated TDEC but similar effects were not seen in MYSEC following 48-hour treatment. Proliferation assays were utilized and TDEC demonstrated decreased proliferation compared to normal endothelial cells at 48 h. To determine whether or not the VDR signaling was impaired in MYSEC, we performed the 24-hydroxylase (CYP24) promoter-luciferase reporter assay. CYP24 is a key enzyme involved in the breakdown of vitamin D. VDR signaling was intact in both cell types and calcitriol induced CYP24 mRNA expression in MYSEC but not in TDEC. Taken together, despite similar levels of VDR expression and intact signaling in both cell types, calcitriol selectively inhibits proliferation and induces apoptosis in TDEC with no effect on MYSEC. Thus calcitriol exerts differential effects on TDEC compared to normal cells.