Circulating gastrin is increased in hemochromatosis

AbstractGastric acid production is important in intestinal iron absorption. The peptide hormone gastrin exists in both amidated and non-amidated forms, which stimulate and potentiate gastric acid secretion, respectively. Since non-amidated gastrins require ferric ions for biological activity in vitro, this study investigated the connection between iron status and gastrin by measurement of circulating gastrin concentrations in mice and humans with hemochromatosis. Gastrin concentrations are increased in the plasma and gastric mucosa of Hfe−/− mice, and in the sera of humans with HFE-related hemochromatosis. The discovery of a relationship between iron status and circulating gastrin concentrations opens a new perspective on the mechanisms of iron homeostasis

Interrelationships between circulating gastrin and iron status in mice and humans

The observations that the peptide hormone gastrin interacts with transferrin in vitro and that circulating gastrin concentrations are increased in the iron-loading disorder hemochromatosis suggest a possible link between gastrin and iron homeostasis. This study tested the hypothesis that gastrin and iron status are interrelated by measurement of iron homeostasis in mice and humans with abnormal circulating gastrin concentrations. Intestinal iron absorption was determined by 59Fe uptake following oral gavage, and concentrations of duodenal divalent metal transporter-1 (DMT-1) and hepatic hepcidin mRNAs were determined by quantitative real-time PCR in agastrinemic (GasKO), hypergastrinemic cholecystokinin 2 receptor-deficient (CCK2RKO), or wild-type mice. Iron status was measured by standard methods in the same mice and in hypergastrinemic humans with multiple endocrine neoplasia type 1 (MEN-1). Iron absorption was increased sixfold and DMT-1 mRNA concentration fourfold, and transferrin saturation was reduced 0.8-fold and hepcidin mRNA expression 0.5-fold in juvenile GasKO mice compared with age-matched wild-type mice. In mature mice, few differences were observed between the strains. Juvenile CCK2RKO mice were hypergastrinemic and had a 5.4-fold higher DMT-1 mRNA concentration than wild-type mice without any increase in iron absorption. In contrast to juvenile GasKO mice, juvenile CCK2RKO mice had a 1.5-fold greater transferrin saturation, which was reflected in a twofold increase in liver iron deposition at maturity compared with wild-type mice. The correlation between transferrin saturation and circulating gastrin concentration observed in mutant mice was also observed in human patients with MEN, in whom hypergastrinemia correlated positively (P = 0.004) with an increased transferrin saturation. Our data indicate that, in juvenile animals when iron demand is high, circulating gastrin concentrations may alter iron status by a CCK2R-independent mechanism

Detection and Cultivation of Soil Verrucomicrobia

Only one isolate each of the class “Spartobacteria” (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class “Spartobacteria” and were related to “Chthoniobacter flavus.” Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class “Spartobacteria” were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class “Spartobacteria” were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class “Spartobacteria” was inhibited by other culturable bacteria

Stimulation of proliferation in the colorectal mucosa by gastrin precursors is blocked by desferrioxamine

Precursors of the peptide hormone gastrin stimulate proliferation in the colorectal mucosa and promote the development of colorectal carcinoma. Gastrins bind two ferric ions selectively and with high affinity, and the biological activity of glycine-extended gastrin (Ggly) in vitro is dependent on the presence of ferric ions. The aim of the present study was to determine whether or not iron is required for biological activity of progastrin and Ggly in vivo. Rats that had undergone a colostomy were infused with Ggly, and proliferation was measured in the defunctioned rectal mucosa. Proliferation was also measured in the colonic mucosa of hGAS and MTI-Ggly mice, which, by definition, overexpress progastrin and Ggly, respectively. The requirement for iron was assessed by thrice-weekly injection of the chelating agent desferrioxamine (DFO). The proliferation index in the defunctioned rectal mucosa was significantly increased in the Ggly-infused rats, and the increase was significantly reduced after treatment with DFO. Treatment with DFO significantly reduced the crypt height and proliferation index in the colonic mucosa of hGAS and MTI-Ggly mice but had no effect on the same variables in wild-type mice. These observations are consistent with the hypothesis that the biological activity of progastrin and Ggly in vivo is dependent on the presence of ferric ions and further suggest that chelating agents may block the stimulatory effects of gastrin precursors in the development of colorectal carcinoma

Knockdown of HIF1α expression in PC3 cells reduced both survival after cytotoxic treatments and migration rate.

<p>(A) HIF1α concentrations were reduced in 2 separate clones of PC3 cells following stable expression of HIF1α shRNA as assessed by Western blot. Values are the mean ± SEM of at least three separate experiments and are expressed as a percentage of wild-type PC3 cells. *, P<0.05 versus wild-type PC3 cells. (B) The survival of PC3 cells after exposure to oxidative stress (hydrogen peroxide (H₂O₂)) or chemotoxicity (5-fluorouracil (5-FU) for 24 hours was reduced following HIF1α knockdown compared to scrambled control vector-transfected PC3 cells. Values are the mean ± SEM of at least three separate experiments and are expressed as a percentage of untreated scrambled control vector-transfected PC3 cells. #, P<0.05 versus control. (C) HIF1α protein expression in PC3 cells transfected with control shRNA after treatment with 1% O₂, 300 µM CoCl₂, 100 µM H₂O₂, and 15 µM 5-FU. Cell lysates were electrophoresed on SDS-polyacrylamide gels and blotted with HIF1α antibody. GAPDH expression was used as loading control. The Western blots shown are representative of at least three separate experiments. Band densities were determined by densitometric analysis of HIF1α/GAPDH and are presented relative to the value for untreated cells. Data represent mean ± SEM; * p<0.05 vs. untreated PC3 cells. (D) Rates of migration/invasion in the HIF1α knockdown PC3 cells were reduced compared to the scrambled control vector-transfected PC3 cells as assessed by Transwell assay. Values are the mean ± SEM of at least three separate experiments and are expressed as a percentage of untreated scrambled control vector transfected PC3 cells. *, P<0.05 versus control. (E) Induction of HIF1α in LNCaP cells by hypoxia (dark grey bars) or by cobalt chloride (light grey bars) increased survival after exposure to oxidative stress with H₂O₂ or chemotoxicity with 5-FU for 24 hours when compared to control LNCaP cells (black bars). Values are the mean ± SEM of at least three separate treatments and are expressed as a percentage of the untreated LNCaP control. #, P<0.05 versus treated LNCaP cells. *, P<0.05 versus LNCaP cells treated with 1% O₂ and 5-FU. (F) HIF1α protein expression in LNCaP cells treated with 1% O₂ and 300 µM CoCl₂ in combination with either 100 µM H₂O₂ or 15 µM 5-FU. Cell lysates were electrophoresed on SDS-polyacrylamide gels and blotted with HIF1α antibody. GAPDH expression was used as loading control. The Western blots shown are representative of at least three separate experiments. Band densities were determined by densitometric analysis of HIF1α/GAPDH and are presented relative to the value for normoxic cells undergoing the same treatment. Data represent mean ± SEM; * p<0.05 vs. untreated control, 100 µM H₂O₂ or 15 µM 5-FU treated LNCaP cells.</p

Univariate and Multivariate Cox Regression analysis of the development of metastatic PC from the time of surgery and CRPC, prostate cancer specific death after starting androgen deprivation therapy.

a<p>13 patients excluded due to incomplete metastasis related data.</p>†<p>Cox regression with Firth’s penalized maximum likelihood method. CI denotes confidence interval.</p>‡<p>This group served as the reference group in the Cox regression analysis.</p>*<p>Pre-interventional PSA was defined as PSA immediately prior to obtaining the tissue sample.</p

Basal HIF1α protein expression, proliferation rates and migration/invasion rates in human PC cell lines. (

<p>A) Basal HIF1α protein concentrations in the human PC cell lines LNCaP, DU145 and PC3 under normoxic conditions were analyzed by Western blot. (B) Proliferation was assayed by cell counting after 24 and 48 hours. (C) Migration/invasion rates were measured by Transwell assays at 24 hours. Values in (A) and (C) are expressed as the fold increase compared to LNCaP cells, while the values in (B) are expressed as a percentage of the time 0 value. All values are the mean ± SEM of at least three separate treatments. (D) Survival rates of PC cells exposed to cytotoxic conditions. The survival of PC3 cells (which have higher basal HIF1α protein) when exposed to oxidative stress with hydrogen peroxide (H₂O₂) or chemotoxicity with 5-fluorouracil (5-FU) was compared to the survival of LNCaP cells (which have lower HIF1α expression). Survival was assessed by counting cell numbers at 24 hours. Values are expressed as a percentage of the untreated control and are the mean ± SEM of at least three separate treatments. #, P<0.05 versus treated LNCaP cells.</p