HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Serrated route to colorectal cancer: back street or super highway?

Morphological and molecular studies are beginning to distinguish separate evolutionary pathways for colorectal cancer, The serrated pathway encompassing hyperplastic aberrant crypt foci, hyperplastic polyps. mixed polyps, and serrated adenoma is increasingly being linked with genetic alterations, including DNA methylation, DNA microsatellite instability, Ii-ras mutation, and loss of chromosome Ip, The importance of the serrated pathway has been underestimated in terms of its frequency and potential for rapid progression, Copyright (C) 2001 John Wiley & Sons, Ltd.

Hyperplastic polyposis - Association with colorectal cancer

Hyperplastic polyposis is a loosely defined syndrome initially thought not to confer a clinically important predisposition to colorectal cancer. The aim of the current study was to examine the clinical, histologic, and molecular features of a prospective series of cases meeting a strict definition of the condition. Twelve patients were identified, seven of whom had developed colorectal cancer. Most polyps were hyperplastic, but 11 patients also had polyps containing dysplasia as either serrated adenomas. mixed polyps, or traditional adenomas. The mean percentage of dysplastic polyps in patients with cancer was 35%, and in patients without cancer, 11%(p < 0.05). Microsatellite instability (MSI) was present in 3 of 47 hyperplastic polyps and two of right serrated adenomas. Kras was mutated in 8 of 47 hyperplastic polyps and two of eight serrated adenomas. No polyps showed loss of heterozygosity of chromosomes 5q, 1p, or 18q. Two of seven cancers showed a high level of MSI. It is concluded that hyperplastic polyposis is associated with a high risk of colorectal cancer. Hyperplastic polyps are the dominant type of polyp, but most cases have some dysplastic epithelium. A higher proportion of dysplastic polyps is associated with increased cancer risk. Clonal generic changes are observed in some hyperplastic polyps and serrated adenomas.

Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings

High-level microsatellite instability (AISI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting In the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might he discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of bMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta -catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than RNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta -catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all RN-PCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.

Methylation of O-6-methylguanine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA microsatellite instability

The significance of low-level DNA microsatellite instability (MSI-L) is not well understood. K-ras mutation is associated with MSI-L colorectal cancer and with the silencing of the DNA repair gene O-6-methylguanine DNA methyltransferase (MGMT) by methylation of its promoter region. MGMT methylation was studied in sporadic colorectal cancers stratified as DNA microsatellite instability-high (n = 23), MSI-L (n = 44), and microsatellite-stable (n = 23). Methylation-specific PCR was used to detect MGMT-promoter hypermethylation in 3 of 23 (13%) microsatellite instability-high, in 28 of 44 (64%) MSI-L, and in 6 of 23 (26%) microsatellite-stable cancers (P = 0.0001). K-ras was mutated in 20 of 29 (69%) methylated MSI-L cancers and in 2 of 15 (13%) unmethylated MSI-L cancers (P = 0.001), indicating a relationship between MGMT-methylation and mutation of K-ras. Loss of nuclear expression of MGMT was demonstrated immunohistochemically in 23 of 31 (74%) cancers with methylated MGMT and in 10 of 49 (20%) cancers with nonmethylated MGMT (P < 0.0001). Loss of expression of MGMT was also demonstrated in 9 of 31 serrated polyps. Silencing of MGMT may predispose to mutation by overwhelming the DNA mismatch repair system and occurs with greatest frequency in MSI-L colorectal cancers.

Hyperplastic polyps of the colorectum - Innocent or guilty?

Hyperplastic polyps have traditionally been regarded as nonneoplastic polyps lacking malignant potential. The demonstration of genetic alterations within these lesions indicates an underlying neoplastic cause. There is evidence that hyperplastic polyps are heterogeneous. Most are innocuous, but subsets may have malignant potential. Risk factors for neoplastic progression include multiple, large, and proximally located polyps. Aberrant methylation resulting in the silencing of cancer genes may be an important underlying mechanism, particularly in pathways progressing to tumors with DNA microsatellite instability. Lesions intermediate between hyperplastic polyp and cancer include admired polyps and serrated adenomas. Currently, pathologists have different thresholds for diagnosing serrated adenomas, including the distinction from large hyperplastic polyps. Reasons for over looking this pathway in the past may include rapid tumor progression and the fact that proximally located hyperplastic polyps may be flat and not especially numerous. Management of the serrated pathway of colorectal neoplasia may require novel approaches to screening, early detection, and prevention.

Proliferation, apoptosis, and survival in high-level microsatellite instability sporadic colorectal cancer

Sporadic colorectal cancer (CRC) characterized by high-level DNA microsatellite instability (MSI-H) has a favorable prognosis. The reason for this MSI-H survival advantage is not known. The aim of this study was to correlate proliferation, apoptosis, and prognosis in CRC stratified by MSI status. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody in a selected series of 100 sporadic colorectal cancers classified according to the level of MSI as 31 MSI-H, 29 MSI-Low (MSI-L), and 40 microsatellite stable (MISS). The Ki-67 index was significantly higher in MSI-H cancers (P < 0.0001) in which the PI was 90.1 1.2% (mean +/- SE) compared with 69.5 +/- 3.1 % and 69.5 +/- 2.3 % in MSI-L and MSS subgroups, respectively. There was a positive linear correlation between the apoptotic index (AI) and PI (r = 0.51; P < 0.001), with MSI-H cancers demonstrating an increased AI:PI ratio indicative of a lower index of cell production. A high PI showed a trend toward predicting improved survival within MSI-H cancers (P = 0.09) but did not predict survival in MSI-L or MSS cancers. The Al was not associated with survival in any MSI subgroup. In conclusion, this is the first study to show that sporadic MSI-H cancers are characterized by a higher AL:PI ratio and increased proliferative activity compared with MSI-L and MSS cancers, and that an elevated PI may confer a survival advantage within the MSI-H subset.

Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status

Background-The presence of high level DNA microsatellite instability (MSI-H) in colorectal cancer is associated with an improved prognosis, as is the presence of tumour infiltrating lymphocytes (TILs). It is not clear if TILs contribute directly to the survival advantage associated with MSI-H cancers through activation of an antitumour immune response. Aims-To correlate TIL and apoptosis rates in colorectal cancer stratified by MSI status. Methods-The distribution of TILs was characterised and quantified in a selected series of 102 sporadic colorectal cancers classified according to levels of MSI as 32 MSI-H, 30 MSI-low (MSI-L), and 40 microsatellite stable (MSS). Archival blocks were immunostained using the T cell markers CD3 and CD8, and the B cell marker CD20. Apoptosis of malignant epithelial cells was quantified by immunohistochemistry with the M30 CytoDEATH antibody. Results-Positive staining with anti-CD3 and negative staining with anti-CD20 identified virtually all TILs as T cells. The majority of CD3(+) TILs (>75%) also stained with anti-CDS. TILs were most abundant in MSI-H colorectal cancers in which 23/32 (72%) scored as TIL positive. Only 5/40 (12.5%) MSS tumours and 9/30 (30%) MSI-L cancers were TIL positive (p

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias

KCNQ1 (K(V)LQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study. the properties and regulation of the cloned sK(V)LQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (

Regulation and properties of KCNQ1 (K(v)LQT1) and impact of the cystic fibrosis transmembrane conductance regulator

The K+ channel KCNQ1 (K(V)LQT1) is a voltage-gated K+ channel, coexpressed with regulatory subunits such as KCNE1 (IsK, mink) or KCNE3, depending on the tissue examined. Here, we investigate regulation and properties of human and rat KCNQ1 and the impact of regulators such as KCNE1 and KCNE3. Because the cystic fibrosis transmembrane conductance regulator (CFTR) has also been suggested to regulate KCNQ1 channels we studied the effects of CFTR on KCNQ1 in Xenopus oocytes, Expression of both human and rat KCNQ1 induced time dependent K+ currents that were sensitive to Ba2+ and 293B. Coexpression with KCNE1 delayed voltage activation, while coexpression with KCNE3 accelerated current activation. KCNQ1 currents were activated by an increase in intracellular cAMP, independent of coexpression with KCNE1 or KCNE3. cAMP dependent activation was abolished in N-terminal truncated hKCNQ1 but was still detectable after deletion of a single PKA phosphorylation motif. In the presence but not in the absence of KCNE1 or KCNE3, K+ currents were activated by the Ca2+ ionophore ionomycin. Coexpression of CFTR with either human or rat KCNQ1 had no impact on regulation of KCNQ1 K+ currents by cAMP but slightly shifted the concentration response curve for 293B. Thus, KCNQ1 expressed in Xenopus oocytes is regulated by cAMP and Ca2+ but is not affected by CFTR.

Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Expression and function of colonic epithelial K(v)LQT1 K+ channels

1. K(V)LQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential Defects in ion channels have been demonstrated in cardiac arrhythmia. This channel is inhibited potently by the chromanol 293B, The same compound has been shown to block cAMP-dependent electrolyte secretion in rat and human colon, Therefore, it was suggested that a K+ channel similar to K(V)LQT1 is expressed in the colonic epithelium. 2, In the present paper, expression of K(V)LQT1 and its function in colonic epithelial cells is described. Reverse transcription-polymerase chain reaction analysis of rat colonic mucosa demonstrated expression of K(V)LQT1 in both crypt cells and surface epithelium. When expressed in Xenopus oocytes, K(V)LQT1 induced a typical delayed activated K+ current. 3, As demonstrated, the channel activity could be further activated by increases in intracellular cAMP. These and other data support the concept that K(V)LQT1 is forming a component of the basolateral cAMP-activated Kf conductance in the colonic epithelium.

A zebrafish homologue of deleted in colorectal cancer (zdcc) is expressed in the first neuronal clusters of the developing brain

DCC (deleted in colon cancer), Neogenin and UNC-5 are all members of the immunoglobulin superfamily of transmembrane receptors which are believed to play a role in axon guidance by binding to their ligands, the Netrin/UNC-40 family of secreted molecules (Cell. Mol. Life Sci. 56 (1999) 62; Curr. Opin. Genet. Dev. 7 (1997) 87). Although zebrafish homologues of the Netrin family of secreted molecules have been reported, to date there has been no published description of zebrafish DCC homologues (Mol. Cell. Neurosci. 9 (1997) 293., Mol. Cell. Neurosci. I I ( 1998) 194; Mech. Dev. 62 (1997) 147). We report here the expression pattern of a zebrafish dcc (zdcc) homologue during the initial period of neurogenesis and axon tract formation within the developing central nervous system. Between 12 and 33 h post-fertilisation zdcc is expressed in a dynamic spatiotemporal pattern in all major subdivisions of the central nervous system. Double-labelling for zdcc and the post-mitotic neuronal marker HNK-1 revealed that subpopulations of neurons within the first nuclei of the zebrafish brain express zdcc. These results support our previous observation that patterning of neuronal clusters in the zebrafish brain occurs early in development (Dev. Bioi, 229 (2001) 271). (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Does 'imprinting' with low prenatal vitamin D contribute to the risk of various adult disorders?

Hypovitaminosis D is a candidate risk-modifying factor for a diverse range of disorders apart from rickets and osteoporosis. Based on epidemiology, and on in vitro and animal experiment, vitamin D has been linked to multiple sclerosis, certain cancers (prostate, breast and colorectal), insulin-dependent diabetes mellitus and schizophrenia. I hypothesise that low pre- and perinatal vitamin D levels imprint on the functional characteristics of various tissues throughout the body, leaving the affected individual at increased risk of developing a range of adult-onset disorders. The hypothesis draws from recent advances in our understanding of the early origin of adult disease and proposes a 'critical window' during which vitamin D levels may have a persisting impact on adult health outcomes. Methods to test the hypothesis are outlined. If correct, the hypothesis has important implications for public health. Careful attention to maternal vitamin D status could translate into diverse improvements in health outcomes for the following generation. (C) 2001 Harcourt Publishers Ltd.

Young patients with colorectal cancer: How do they fare?

Background: Younger patients with colorectal cancer (CRC) have long been thought to have a poorer prognosis than older patients. Recent overseas reports, however, have disputed this. The aim of the present study was to conduct a review of data on patients with colorectal cancer collected over a 29-year period at Princess Alexandra Hospital (PAH) to ascertain the outcome of a younger subset of patients at this hospital. Methods: The PAH Colorectal Project records on 2495 patients with malignancies of the colon, rectum and anus who were treated and followed since 1971, were analysed to determine clinical presentation, treatment and outcome. A group of 61 patients with colo-rectal adenocarcinoma was identified who were aged less than 40 years at presentation. Their clinical data were then compared with the larger group of older patients. Results: There were 30 male and 31 female patients in the younger group. A positive family history was the most consistent risk factor, present in 34% of patients. Despite this, only one patient out of 61 had been diagnosed as a result of a screening programme. The Australian Clinico-Pathological Stage (ACPS), histology and distribution of tumours corresponded to that of the older patients. The overall 5-year survival among younger patients was 53%. The 5-year survival rates in younger patients were better than that for older patients for ACPS A and B, reaching statistical significance for both of these stages. Conclusions: Our results indicate that younger patients with colorectal cancer have the potential to do just as well as older ones. With the influence of a family history of colorectal cancer being very apparent in this group, greater emphasis should be placed on an adequate screening programme for them.