Estrogen receptor alpha gene polymorphisms are associated with changes in bone remodeling markers and treatment response to estrogen.

OBJECTIVES: Association studies between estrogen receptor alpha (ERalpha) gene polymorphisms and bone mineral density (BMD) have yielded inconsistent results. In the present study we evaluated the influence of XbaI and PvuII ERalpha gene polymorphisms on BMD, biochemical markers, rates of bone loss and the response to estrogen/hormone therapy (ET/HT) in elderly postmenopausal women. METHODS: At baseline, we measured the association between ERalpha genotypes and BMD and biochemical markers in 489 elderly women, mean age 71 +/- 3 years. In the longitudinal study, the changes in the same measures were determined in 96 women on placebo and in 79 women receiving the ET/HT for 3 years. The XbaI and PvuII ERalpha polymorphisms were determined by polymerase chain reaction (PCR). BMD measurements for spine, femoral neck and total body were performed by DEXA, and biochemical indices were measured by standard methods. RESULTS: Neither the PvuII nor the XbaI ERalpha gene polymorphisms were associated with baseline BMD and biochemical indices. In the longitudinal study, there were trends for higher bone loss in the placebo group in the genotypes pp or xx compared to PP or XX genotypes, but the changes were not significant. However, the changes in the bone markers were significantly (p < 0.05) higher in genotype group pp compared to PP (serum osteocalcin, 4.9 +/- 7.0% versus -13.4 +/- 6.7%; urine NTx:Cr ratio, 32.3+/-10.3% versus -2.5 +/- 10.3%) or xx compared to XX (serum osteocalcin, 7.5 +/- 6.4% versus -15.6+/-7.3%; urine NTx:Cr ratio, 39.4 +/- 9.2% versus -8.84+/-10.7%). At the end of 3 years, the mean urine NTx:Cr ratio was 78.7 +/- 9.0 versus 44.6 +/- 4.9 in pp versus PP (p < 0.05) and 75.5 +/- 10.7 versus 48.7 +/- 5.4 in xx versus XX (p < 0.05) genotypes. The response in total body BMD to ET/HT treatment was significantly higher in women with the PP genotype compared to pp genotype (2.48 +/- 0.55% versus 0.66 +/- 0.46%). Similar trends were seen at other skeletal sites for both XX and PP compared to pp and xx genotypes. CONCLUSION: Women with ERalpha, PP and XX genotypes have lower bone remodeling, lower rates of bone loss and benefit more from hormone therapy.

Association between Vitamin D receptor polymorphisms and the rate of bone loss in elderly women-importance of adjusting for dietary and lifestyle factors.

The association between the restriction length polymorphisms of the Vitamin D receptor (VDR) gene and the bone mineral density (BMD) or the rate of bone loss is still under debate. In a longitudinal study of untreated postmenopausal elderly women, we evaluated the relationship between the VDR gene polymorphisms (BsmI, TaqI, ApaI, and FokI) and the rate of bone loss over a 3-year period. We also examined the effect of adjustments for dietary and lifestyle factors on these associations. Before adjustments, the rate of femoral neck bone loss was - 3.76 +/- 1.58% in women with BB genotype and 0.45 +/- 0.65% in women with bb genotype, which was not significantly different. Upon adjustment for dietary and lifestyle factors, statistically significant (P = 0.03) bone loss was observed at femoral neck in women with BB genotype (- 3.66 +/- 2.44%) compared to that of bb genotype (2.39 +/- 1.32%). Similar results were observed with TaqI genotypes. The rates of bone loss at other skeletal sites were not different between VDR genotypes defined by BsmI and TaqI. VDR gene polymorphisms defined by ApaI and FokI were not related to the rate of bone loss.

Cloning and tissue distribution of two Na+/H+ exchangers from the Malpighian tubules of Aedes aegypti.

A Na(+)/H(+) exchanger (NHE) on the apical membrane of mosquito Malpighian tubule (MT) cells is believed to participate in the blood-feeding mosquito's vital secretion of Na(+) and fluid. This study presents the molecular cloning, primary structure, and tissue distribution of two cDNAs encoding Aedes aegypti mosquito MT NHEs. The cDNA sequences were obtained from mosquito MT total RNA using reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE). The two sequences encode proteins of 678 and 1,179 amino acids with calculated molecular weights of 74,473 and 130,276, respectively. When comparing the 678 amino acid protein to the first 678 amino acids of the other protein, the two clones show 98% identity to each other. They also exhibit high identity to Drosophila melanogaster NHEs. Hydropathy analysis reveals that while both clones have 10-13 transmembrane segments, the 1,179 amino acid protein has an extensive carboxy terminus while the 678 amino acid protein has an extremely short carboxy terminus. RT-PCR analysis shows that both clones are expressed in the mosquito Malpighian tubules at the larval and pupal stages, in addition to the adult stage before and after blood-feeding. Expression of both clones was also detected in adult mosquito ovaries, midguts, and hindguts.

Receptors and ligands for autocrine growth pathways are up-regulated when pancreatic cancer cells are adapted to serum-free culture.

Overexpression of autocrine growth factors and their receptors has been reported in many human cancers. The study of autocrine-regulated pathways using in vitro culture systems can be hindered by the presence of fetal bovine serum in culture medium. A human pancreatic cancer cell line (HPAF) was slowly weaned from its dependence on fetal bovine serum and subsequently maintained in serum-free conditions. Growth factor secretion studies showed that production of autocrine growth factors such as transforming growth factor alpha, gastrin-releasing peptide, and insulin-like growth factor I from weaned cells increased three times compared with nonweaned cells (p < 0.01). The epidermal growth factor and gastrin-releasing peptide receptor densities were also increased in weaned cells (2 times and 2.5 times, respectively, p < 0.05). The proliferation of weaned cells cultured continuously in the same medium was significantly greater than of nonweaned cells (p < 0.05). Collectively, these data indicate that weaned pancreatic cancer cells can proliferate in the absence of serum by up-regulating autocrine pathways.

Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions.

In-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently (HPAF, CAPAN-2, PANC-1 or SW1990) or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c- jun and c- fos mRNA levels were significantly elevated in CAPAN-2 cells cultured continuously in serum-free medium. Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells.

The intracellular mechanism of insulin resistance in pancreatic cancer patients.

The diabetes that frequently occurs in pancreatic cancer patients is characterized by profound peripheral insulin resistance. The intracellular mechanism of this insulin resistance was investigated in skeletal muscle biopsies from pancreatic cancer patients with or without diabetes and control subjects. Insulin receptor (IR) binding, tyrosine kinase activity, IR messenger RNA (mRNA), IR substrate-1 content, GLUT-4, and GLUT-4 mRNA content were all normal in pancreatic cancer patients. In contrast, multiple defects in glycogen synthesis were found in pancreatic cancer patients, especially in those with diabetes. Glycogen synthase I activity, total activity, and mRNA levels were significantly decreased in pancreatic cancer patients compared with controls. The fractional velocity of glycogen synthase was decreased only in the diabetic pancreatic cancer group. Glycogen phosphorylase a and b activities were increased in diabetic pancreatic cancer patients, but glycogen phosphorylase mRNA levels were not significantly different. The insulin resistance associated with pancreatic cancer is associated with a post-IR defect, which impairs skeletal muscle glycogen synthesis and glycogen storage.

Microsatellite instability and expression of MLH1 and MSH2 in normal and malignant endometrial and ovarian epithelium in hereditary nonpolyposis colorectal cancer family members.

Mismatch repair deficiency is a characteristic molecular finding in hereditary nonpolyposis colorectal cancer (HNPCC), and has been demonstrated in both colorectal cancers and benign adenomas. Endometrial and ovarian cancers are common extracolonic tumors in this syndrome; however, few studies have investigated whether genetic changes occur in histologically normal endometrial and ovarian epithelia from HNPCC family members. If early genetic changes exist, they might be used as molecular markers to detect susceptibility to endometrial and ovarian cancers. In this study, we analyzed microsatellite instability (MSI) and MLH1 and MSH2 immunohistochemical expression in 20 histologically normal epithelia (12 endometrial and 8 ovarian) and 8 cancers (4 endometrial and 4 ovarian) obtained from 20 individuals representing 7 unrelated HNPCC families. While MSI was observed in endometrial (75%) and ovarian (100%) cancers, no case was determined to exhibit MSI in histologically normal epithelia of the endometrium or ovary. Similarly, in immunohistochemical expressions for MLH1 and MSH2, histologically normal epithelia had no genetic changes predisposing to malignancy. In cancer cases, a correlation existed between the expression of MLH1 and MSH2, the presence of germline mutations in the hMLH1 and hMSH2 genes, and the presence of tumor MSI. These data suggest that MSI and MLH1 and MSH2 expression are not useful biomarkers for the early detection of endometrial and ovarian malignancy in cancer-unaffected HNPCC germline mutation carriers. Further studies of other genetic changes in normal and premalignant precursor lesions are needed.

Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by (3)H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.

A century of progress in hereditary nonpolyposis colorectal cancer (Lynch syndrome).

One of the earliest references to heredity in colorectal cancer dates to Aldred Warthin's now-famous recollection of his seamstress' distress regarding "cancer excess" in her family history. Her prediction of an early demise secondary to cancer of the female organs, colon, or stomach proved true. The slow, arduous investigation that ensued followed a tortuous route of nearly eight decades before the implications of such family histories were widely acknowledged through the designation of hereditary nonpolyposis colorectal cancer or Lynch Syndrome Variants I and II. The story of hereditary nonpolyposis colorectal cancer is one of chance meetings, the selfless sharing of information, perseverance in the face of adversity, meticulous scientific documentation, and ultimate vindication by a scientific process that yielded molecular genetic evidence through the identification of the culprit mutations (hMSH2, hMLH1, hPMS2, and hMSH6). Our purpose is to provide a brief outline of the course charted by the study of the genetics of hereditary nonpolyposis colorectal cancer. This should be of particular interest to the readers of this Journal as we celebrate 100 years of dedication to the diagnosis and treatment of diseases of the colon, rectum, and anus through the efforts of The American Society of Colon and Rectal Surgeons.

Effect of vitamin D receptor genotypes on calcium absorption, duodenal vitamin D receptor concentration, and serum 1,25 dihydroxyvitamin D levels in normal women.

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)2D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25-35 years and 43 elderly women aged 65-83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1, 25(OH)2D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism.

Bombesin may stimulate proliferation of human pancreatic cancer cells through an autocrine pathway.

Bombesin is trophic to normal pancreas and acinar cell adenocarcinoma, but its effects on ductal cell tumors are undetermined. The autocrine growth effects of bombesin on well-differentiated (HPAF, CD11) and poorly differentiated (CD18, PANC-1) human ductal pancreatic cancer cell lines were investigated. Receptor binding of labeled bombesin was measured in a whole-cell microplate assay. Bombesin production was measured by radioimmunoassay. Proliferative responses were quantified using the MTT assay. Messenger RNA for bombesin and its receptor were identified by primer extension analysis. A single class of high-affinity binding sites was detected on HPAF and CD18 cells. Similar affinities and high receptor densities were found on the 2 cell lines. Bombesin was secreted by all 4 cell lines during 24-hr culture in serum-free media, and its recovery was enhanced in the presence of protease inhibitors. Primer extension analysis demonstrated the presence of mRNA for both bombesin and its receptor in HPAF, CD18, CD11 and PANC-1 cells, even though no functional receptor was found in the latter 2 lines. Bombesin significantly stimulated the proliferation of HPAF and CD18 cells. This trophic effect was inhibited by the specific bombesin antagonist RC-3095. Bombesin may act as an autocrine growth factor in some human pancreatic cancer cell lines. Furthermore, other cell lines transcribe mRNA for bombesin receptors but have no functional bombesin receptors, suggesting a genetic or post-translational change in the receptor for these cells. Bombesin may be involved as a growth factor in the development of pancreatic ductal adenocarcinoma in humans. This possible autocrine growth pathway may provide an avenue for therapeutic intervention in this malignant disease.

Mechanism of developmental regulation of alpha pi, the chicken embryonic alpha-globin gene.

The chicken alpha pi-globin gene is expressed during development only in the primitive erythrocyte lineage and not in the definitive lineage. We show that stage-specific expression is maintained when plasmids containing the alpha pi promoter are transfected into primitive and definitive lineage primary erythroid cells and that the information contained in the promoter is sufficient to confer this specificity. Detailed analysis of binding sites in the promoter for trans-acting factors, together with studies of the effects of mutagenesis on expression, reveals that the factors critical to stage-specific expression are all present in both primitive and definitive lineages, but at various concentrations. We identify three proteins, an NF1 family member, a Y-box factor, and an Sp1-like factor, which interact to stimulate or inhibit transcription. We propose that the concentration-dependent action of these factors, together with the general erythroid factor GATA-1, is responsible for the stage-specific expression of the alpha pi-globin gene.

Identification and characterization of a chicken alpha-globin enhancer.

We identify and describe the properties of an enhancer within the chicken alpha-globin gene cluster. This cluster consists of one gene (pi) expressed only in primitive erythrocytes and two (alpha A and alpha D) expressed in both primitive and definitive cell lineages. The genes are linked together in the order 5'-pi-alpha D-alpha A-3' and occupy a region about 10 kilobase pairs long. The enhancer is located at the 3' end of the cluster, about 750 base pairs 3' to the alpha A translation stop site. When assayed by transfection into either primitive or definitive primary chicken erythrocytes, this element stimulated expression from plasmids containing the alpha D- or alpha A-globulin gene promoters. Except for sites in the alpha-globin promoters, no other stimulatory activity was observed in DNA taken from other regions of the alpha-globin locus. Moderate resolution DNase I hypersensitivity studies as well as DNase I footprinting revealed three regions of protein binding, each containing a similar core DNA sequence within the enhancer element. Gel mobility shift studies demonstrated that all three regions bind the recently identified erythrocyte-specific factor, EryfI, which has binding sites in the regulatory regions of all chicken globin genes. Our data suggest that the enhancer we have identified may act in vivo only on the alpha A gene; expression of the alpha D gene is affected by another EryfI site located in the alpha D promoter. Such a mechanism would be consistent with the observed relative abundances of alpha A- and alpha D-globin in vivo. The simplicity of these regulatory elements may reflect the limited repertoire of expression of these genes during development.

Assembly of RNA polymerase II preinitiation complexes before assembly of nucleosomes allows efficient initiation of transcription on nucleosomal templates.

We have previously shown that assembly of nucleosomes on the DNA template blocks transcription initiation by RNA polymerase II in vitro. In the studies reported here, we demonstrate that assembly of a complete RNA polymerase II preinitiation complex before nucleosome assembly results in nucleosomal templates which support initiation in vitro as efficiently as naked DNA. Control experiments prove that our observations are not the result of slow displacement of nucleosomes by the transcription machinery during chromatin assembly, nor are they an artifact of inefficient nucleosome deposition on templates already bearing an RNA polymerase. Thus, the RNA polymerase II preinitiation complex appears to be resistant to disruption by subsequent nucleosome assembly.

The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro.

RNA was synthesized in vitro using HeLa cell nuclear extracts and circular DNA templates onto which varying numbers of nucleosomes had been reconstituted with Xenopus oocyte extracts. We found that fully reconstituted templates supported no specific initiation by RNA polymerase II; however, DNA exposed to the reconstitution extracts under conditions which did not allow nucleosome deposition was transcribed normally. A set of successively less reconstituted templates was also transcribed. No initiation occurred on reconstitutes with more than two-thirds of the physiological nucleosome density; reconstitutes with less than one-third of the physiological nucleosome density were transcribed as efficiently as naked DNA.