Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNA clones for calretinin, a member of the troponin-C family of calcium-binding proteins, were isolated from a cDNA library of the human colon carcinoma cell line WiDr. Sequence analysis revealed two forms of alternatively spliced calretinin mRNAs encoding C-terminally truncated proteins. Exon 7 was either spliced to exon 9 (delta 8) or to exon 10 (delta 8,9); both resulted in a frame shift and a translational stop at the second codon of exon 9 (delta 8), or at codon 15 of exon 10 (delta 8,9), respectively. The presence of delta 8 and delta 8,9 calretinin mRNA in WiDr cells was confirmed using reverse-transcriptase PCR and sequence analysis of the amplicon, as well as by a ribonuclease protection assay. Co115/3 and three other human colon carcinoma cell lines were found, by reverse-transcriptase PCR to also contain delta 8,9 calretinin mRNA. The truncated proteins were able to bind calcium, as evidenced by a calcium blot of the delta 8 form (calretinin-20k) and delta 8,9 form (calretinin-22k) expressed in Escherichia coli. Immunohistochemical staining using an antiserum specific for the novel C-terminus of calretinin-22k confirmed its presence in WiDr, Co115/3 and three additional colon carcinoma cell lines. The fact that alternative splicing of calretinin was found in five different cell lines suggests that alternatively spliced calretinins fulfill a physiological function.
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PMID:Alternative splicing of calretinin mRNA leads to different forms of calretinin. 760 11

The protein tyrosine phosphatase alpha (PTP alpha) mRNA level in paired samples of late stage (Dukes' D) colorectal tumors and adjacent normal colon mucosa was quantified by RNase protection assays. After normalization against 18S RNA or beta-actin mRNA level, a 2-10-fold increase in PTP alpha mRNA was detected in 10 of 14 tumors (approximately 70%) compared to mucosa. In situ hybridization of digoxigenin-labelled antisense PTP alpha RNA to tumor and mucosa sections produced a signal only in neoplastic cells of the tumor sample, consistent with the high increase in PTP alpha mRNA detected by RNase protection assays of some of the tumors. This is the first report suggesting an association of a protein tyrosine phosphatase with colorectal carcinoma. PTP alpha is a receptor-like PTP thought to be involved in regulating cell proliferation. Its oncogenic properties when overexpressed in cultured fibroblasts suggest that PTP alpha overexpression could contribute to the tumorigenic process in colon carcinoma.
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PMID:Increased mRNA expression of the receptor-like protein tyrosine phosphatase alpha in late stage colon carcinomas. 762 35

A gene has been described, Down Regulated in Adenoma (dra), which is expressed in normal colon but is absent in the majority of colon adenomas and adenocarcinomas. However, the function of this protein is unknown. Because of sequence similarity to a recently cloned membrane sulfate transporter in rat liver, the transport function of Dra was examined. We established that dra encodes for a Na(+)-independent transporter for both sulfate and oxalate using microinjected Xenopus oocytes as an assay system. Sulfate transport was sensitive to the anion exchange inhibitor DIDS (4,4'-diisothiocyano-2,2' disulfonic acid stilbene). Using an RNase protection assay, we found that dra mRNA expression is limited to the small intestine and colon in mouse, therefore identifying Dra as an intestine-specific sulfate transporter. dra also had a unique pattern of expression during intestinal development. Northern blot analysis revealed a low level of expression in colon at birth with a marked increase in the first 2 postnatal weeks. In contrast, there was a lower, constant level of expression in small intestine in the postnatal period. Caco-2 cells, a colon carcinoma cell line that differentiates over time in culture, demonstrated a marked induction of dra mRNA as cells progressed from the preconfluent (undifferentiated) to the postconfluent (differentiated) state. These results show that Dra is an intestine-specific Na(+)-independent sulfate transporter that has differential expression during colonic development. This functional characterization provides the foundation for investigation of the role of Dra in intestinal sulfate transport and in the malignant phenotype.
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PMID:The Down regulated in Adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein. 774 40

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.
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PMID:Regulation of autocrine gastrin expression by the TGF alpha autocrine loop. 782 34

Although a great deal is known about the cellular effects of exogenous transforming growth factor-beta (TGF-beta) treatment and the effects of various exogenous agents (including TGF-beta's themselves) on TGF-beta expression, studies of cellular controls for autocrine TGF-beta expression and function have been rare. Since exogenous TGF-beta treatment blocks progression through the cell cycle, it seemed likely that autocrine TGF-beta activity would be induced by growth states in which there was little or no cell division such as confluency or quiescence. Specific TGF-beta 1 or beta 2 neutralizing antibody treatment of a colon carcinoma cell line designated CBS showed that autocrine TGF-beta activity could be demonstrated in quiescent cells but not in preconfluent cells. Studies of kinetics of TGF-beta 1 and beta 2 mRNA levels during the establishment of quiescence revealed a significant increase of both isoforms in quiescent cells. The quiescent cells also secreted three- to fourfold and four-to fivefold higher levels of total (latent plus active) TGF-beta 1 and beta 2 protein in the conditioned media than the confluent cells and preconfluent cells, respectively. There was no detectable active form of either TGF-beta isoform in the conditioned media of preconfluent cells, whereas a significant amount of active TGF-beta 1 and beta 2 was detected in the conditioned media of quiescent cells. Quantitative RNase protection assays were developed to compare the effects of cell crowding vs quiescence on TGF-beta expression. TGF-beta 1 was primarily induced by quiescence. TGF-beta 2 was induced by both quiescence and cell crowding. Increased TGF-beta 1 mRNA levels appeared to be exclusively due to an increase in stability, while increased TGF-beta 2 mRNA levels were due to increased transcription. This growth state-related induction of TGF-beta's was also observed in two other colon carcinoma cell lines. These studies show that TGF-beta 1 and beta 2 are autocrine-negative factors which can be situationally expressed by cells as a function of their growth state. Autocrine expression of the TGF-beta's in this model system appears not to affect exponentially growing cells, but rather to function by maintaining a quiescent state and/or by blocking progression through the cell cycle.
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PMID:Autocrine transforming growth factor-beta 1 and beta 2 expression is increased by cell crowding and quiescence in colon carcinoma cells. 808 24

c-myb expression is an indicator of hemopoietic cell proliferation and its down-regulation occurs as an early event in cellular differentiation in vitro. We have previously demonstrated c-Myb protein expression in normal adult colon and tumor-derived colonic cell lines and have also shown that the proliferation of colon carcinoma cell lines is c-myb dependent. This study used the techniques of RNase protection and immunohistochemistry to define c-myb mRNA and protein expression in the normal adult mouse and mouse embryos, with special focus on the colon. These experiments revealed wide-spread c-myb mRNA expression in mouse embryos including nonhemopoietic tissues, and developmental time course studies revealed alterations in organ-specific c-myb mRNA expression. Immunohistochemical studies confirmed the embryonal expression of c-Myb in concordance with the mRNA data as well as c-Myb expression throughout the length of the adult mouse colonic crypt. In contrast, staining for the proliferating cell nuclear antigen was confined to the lower one third of the crypt. In addition, c-Myb staining extended beyond that of the proliferating cell nuclear antigen within the germinal centers of the spleen. These data suggest that c-myb: (a) has a role in the embryonic development of nonhemopoietic organs; (b) plays a specific role in the biology of the normal adult colonic crypt; and (c) expression is not inextricably linked to proliferation in vivo.
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PMID:Colonic expression of c-myb is initiated in utero and continues throughout adult life. 880 14

Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8- to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation.
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PMID:Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation. 881 6

Lck protein is expressed in some colon carcinoma cell lines but its expression in colon cancer cells in vivo has not been clarified. LCK transcription is regulated from two distinct promoters and initiated exclusively from the downstream promoter in colon carcinoma cell lines in contrast to peripheral lymphocytes. We investigated the expression of the downstream promoter-initiated LCK transcript in 18 colorectal primary cancer and normal mucosae, and two hepatic metastases, using a RNase protection assay with the EcoRI-BglII fragment of human LCK cDNA, YT16. In normal tissues, only traces of the LCK transcript were detected. The expression of the LCK transcript was augmented in 3/18 cancer specimens. The relative level of the LCK transcript in the cancer tissue compared to the average value of normal adjacent tissue was 10-60 in 3 cases, and 3-10 in 7 cases. One hepatic metastasis expressed more LCK message than the primary lesion. Our results indicate that the LCK message is strongly expressed in some colorectal cancers.
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PMID:Augmented expression of LCK message directed from the downstream promoter in human colorectal cancer specimens. 886 6

It is now clearly established that alpha-2 adrenergic receptors can be subdivided in three pharmacological subtypes (alpha-2A, alpha-2B and alpha-2C) encoded by distinct genes (alpha 2C10, alpha 2C2 and alpha 2C4, respectively, in humans). Whereas the study of the regulation of the human alpha-2A adrenergic receptor and of the promoter region of the alpha 2C10 gene has being greatly helped by the availability of the colon carcinoma cell line HT29, the study of the other human receptor subtypes has thus far been limited to homologous desensitization/down-regulation in transfected cells, because of the lack of human cellular models constitutively-expressing alpha-2B or alpha-2C adrenergic receptors. Several human cell lines were thus screened, in an attempt to find such models. Radioligand binding studies with [3H]RX821002 and [3H]MK912, reverse transcription-polymerase chain reactions and RNase mapping experiments with pairs of primers and riboprobes specific for each subtype demonstrated that the hepatoma cell line HepG2 and the neuroblastoma cell line SK-N-MC possess alpha-2 adrenergic receptors of the alpha-2C subtype. However, whereas HepG2 expresses exclusively alpha-2C receptors (55 +/- 7 fmol of [3H]MK912 binding sites/mg of protein), SK-N-MC expresses both alpha-2A and alpha-2C subtypes in fairly similar amounts (20 +/- 8 and 23 +/- 3 fmol of [3H]MK912 binding sites/mg of protein, respectively). The study of the inhibition of 3H-labeled antagonist binding by UK14304 demonstrated that a fraction of the receptor population was coupled to pertussis toxin-sensitive G-proteins, which were identified as Gi2 and Gi3 by immunoblotting. The alpha-2 agonist was, moreover, able to decrease forskolin-stimulated cAMP production by 47% in HepG2 and 23% in SK-N-MC, demonstrating that inhibition of adenylyl cyclase is one of the primary mechanisms of signal transduction in both cell lines. HepG2 and SK-N-MC are the first human cell lines unquestionably shown to natively express alpha-2C adrenergic receptors. The discovery of these two models may be useful for future study of the regulation of alpha 2C4 gene expression in cells of different origins and investigation of the reciprocal regulation of alpha-2A and alpha-2C subtype in single cells.
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PMID:HepG2 and SK-N-MC: two human models to study alpha-2 adrenergic receptors of the alpha-2C subtype. 915 9

The IGFs have been implicated in the development of the intestinal tract. We have studied the human colon carcinoma cell line CaCo-2 to gain more insight into the function of the IGFs in the gut. [125I]IGF-I and -II bound specifically to CaCo-2 cells as measured in competitive binding experiments. The existence of IGF-I receptors was further demonstrated by affinity crosslinking studies using DSS as the crosslinking agent. Western blotting of CaCo-2 cell extracts using an anti IGF-II/M6P receptor antiserum provided additional evidence for the expression of the IGF-II/M6P receptor. In addition, Northern blotting experiments showed specific IGF-I receptor and IGF-II/M6P receptor gene expression in CaCo-2 cells. An 11 kb band was visualized with a 614 bp PstI IGF-I receptor probe on autoradiographs. Hybridization with a 663 bp IGF-II/M6P receptor probe yielded a 9 kb RNA species. Analysis of CaCo-2 cell RNA using solution hybridization/RNase protection assays yielded two protected fragments, approximately 379 bases in length, with a 394 base IGF-I receptor riboprobe and a 250 base protected fragment with a 260 base IGF-II/M6P receptor riboprobe. In a subset of experiments a PstI 700 base fragment of the IGF-I cDNA and a 554 base SalI fragment of the IGF-II cDNA were used for hybridization: no hybridization was detected with the IGF-I probe. However, using the [32P]IGF-II probe bands at 6.0 and 5.0 kb were labeled in Northern blotting experiments. Analysis of CaCo-2 cell RNA using solution hybridization/RNase protection assays yielded a 289 base protected fragment and a faint 534 base species with a 556 base human IGF-II riboprobe. In addition, IGF-II immunoreactivity was measured in CaCo-2 cell-conditioned medium using an IGF-binding protein blocked radioimmunoassay. CaCo-2 cell-conditioned medium contained 5-15 ng/ml IGF-II immunoreactivity. In conclusion, (1) CaCo-2 cells express both IGF-I receptor mRNA and IGF-II/M6P receptor mRNA and contain functional IGF-I receptor and IGF-II/M6P receptor protein. (2) CaCo-2 cells express IGF-II mRNA and secrete IGF-II immunoreactivity. We hypothesize that in human colon carcinoma cells IGF-II could act as an autocrine growth factor or alternatively could serve as a regulatory factor during differentiation.
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PMID:Human colon carcinoma cells (CaCo-2) synthesize IGF-II and express IGF-I receptors and IGF-II/M6P receptors. 939 46


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