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L H Augenlicht

Publications and source records attributed to L H Augenlicht.

At least 19 recordsLinked to original sources
PubMed1995

Presence and instability of repetitive elements in sequences the altered expression of which characterizes risk for colonic cancer.

50C10 and 52H10 are two DNA clones previously reported by us to be overexpressed in human colonic mucosa at high risk for development of colonic tumors. This report presents sequencing data that reveal that these clones contain repetitive Alu elements. Each Alu sequence is associated with a 3'-oligoadenylate [oligo(A)] sequence, which is demonstrated to exhibit instability in human colonic tumors. The oligo(A) sequences only decrease in length, unlike microsatellites, which can either increase or decrease. Rigorous quantitative analysis of the length of the oligo(A) sequence in colonic tumors demonstrates that the standard deviation of the length of the sequence in tumors is a function of the mean length; i.e., as the oligo(A) sequence becomes progressively shorter, the variance increases. Both measurements, therefore, provide a quantitative index of the extent of instability in a tissue. Comparison of instability at the oligo(A) loci defined by 50C10 and 52H10 to instability at a CA microsatellite upstream of the apoD gene, and comparison of an oligo(A) and a CA microsatellite both in the 3' untranslated region of the cyclin D1 mRNA demonstrate that instability in a tumor, when present, is more prominent for the oligo(A) sequences than for the microsatellite (P < 0.0001). This suggests either that the mechanisms that generate oligo(A) instability are more penetrant than those that generate microsatellite instability, or that the instability at oligo(A) sequences takes place earlier in the development of the tumor and is selected for, thus becoming more prominent. These features of oligo(A) instability suggest that they may be uniquely useful in detecting and quantifying instability in tissues. Further, the presence of repetitive sequence elements in loci overexpressed in colonic mucosa at risk may be related to an extensive literature that demonstrates that a variety of repetitive sequences accumulate in the cellular RNA population during carcinogenesis and in tumors. Such RNA sequences could play a mechanistic role in tumor development.

Amino Acid Sequence↗
PubMed1994

Polymorphisms, but lack of mutations or instability, in the promotor region of the mitochondrial genome in human colonic tumors.

The region of the human mitochondrial D-loop has been sequenced from DNA of colonic tumors and paired normal colonic tissue to determine if mutations in the promotors for the heavy or light strands are responsible for the decrease in mitochondrial gene expression present in colonic tumors. No mutations were detected in the colonic tumors, but new polymorphisms, including a sequence analogous to CA microsatellites in genomic DNA, were revealed. These polymorphisms are restricted to positions within the D-loop which are not essential for accurate and efficient in vitro mitochondrial transcription. Thus, these data confirm the boundaries of the functional heavy and light strand promotors determined by in vitro assays. Further, although some of the tumors investigated show genomic microsatellite instability similar to that recently reported for colonic tumors, the CA polymorphic region in the mitochondrial D-loop does not show coincident instability in the tumors. Therefore, as in yeast, there may be both a mitochondrial and a nuclear enzyme responsible for mismatch repair, with only the latter involved in generation of instability in some human colon cancers. In summary, our data do not find any structural alterations in the D-loop region of the human mitochondrial genome encompassing the heavy and light strand promotors which can account for the decreased expression of the mitochondrial genome in colonic tumors.

Base Sequence↗
PubMed1994

Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines.

The architecture of normal colonic mucosa suggest that terminally differentiated epithelial cells near the top of the crypt are extruded into the colonic lumen. Morphological studies have identified apoptotic cells among the differentiated phenotypes near the crypt-lumen interface, suggesting a link between pathways of differentiation, apoptosis, and cellular shedding. We studied these processes in HT29 and SW620 cells and found that compared to adherent cells, those cells which were shed during standard, uninduced culture conditions exhibited nonrandom DNA fragmentation characteristic of apoptosis. Moreover, these apoptotic cells, which accumulate in the media, exhibited a more differentiated phenotype. Because short-chain fatty acids (SCFAs) are natural effectors of colonic cell differentiation in vivo, we investigated the specificity of three 4-carbon atom SCFAs on potentiating differentiation and apoptosis, and thus accumulation of shed cells in the conditioned media, in these colonic carcinoma cell lines. Whereas the unbranched SCFA butyrate induced a more differentiated phenotype and enhanced apoptosis, two derivatives of butyrate, branched isobutyric acid and a nonmetabolizable fluorine-substituted analogue, heptafluorobutyric acid, were ineffective in inducing either differentiation or apoptosis. Thus, potentiated differentiation and apoptosis in colonic carcinoma cells were linked to SCFA structure and, most likely, utilization.

Apoptosis↗
PubMed1994

Interferon-gamma modulates cAMP-induced mucin exocytosis without affecting mucin gene expression in a human colonic goblet cell line.

The regulation of intestinal mucin secretion by cytokines, soluble factors released by mucosal activated immune cells, is so far unknown. The aim of the present study was (1) to investigate the regulatory effects of interferon-gamma on baseline and stimulated mucin secretion elicited by an increase in intracellular cAMP, either a short-term increase (induced by vasoactive intestinal peptide or by forskolin) or a long-term increase (cholera toxin-induced), and (2) to attempt to delineate the site of action of interferon-gamma. The in vitro model used was the human colonic goblet cell line Cl.16E, which has already been shown to respond to physiological secretagogues in terms of mucin secretion. We examined the effects of interferon-gamma 1) on mucin exocytosis, measured as release of [3H]glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels, and 2) on mucin biosynthesis, examined at the RNA level using a cDNA probe directed to the MUC2 mucin gene. We demonstrated that, while interferon-gamma did not alter baseline Cl.16E mucin secretion and MUC2 gene expression, it strongly inhibited the protein kinase A-dependent secretory response to VIP, forskolin, or cholera toxin. However, interferon-gamma had no effect on the protein kinase A-dependent MUC2 over-expression induced by cholera toxin. We thus concluded that the target for interferon-gamma inhibition of cAMP-stimulated Cl.16E mucin secretion is distal to protein kinase A and might be a component of the exocytotic machinery. Together, our results establish interferon-gamma as a pharmacologically powerful tool to specifically inhibit stimulated secretory processes without affecting baseline secretion.

Blotting, Northern↗
PubMed1993

Regulated expression of an intestinal mucin gene in HT29 colonic carcinoma cells.

We have investigated the regulation of the intestinal mucin gene MUC2 in HT29 cells. Surprisingly, sodium butyrate, an effective inducer of aspects of colonic cell differentiation in HT29 cells, fails to induce MUC2 during short-term exposure, despite the fact that it has been used to select stably differentiated clones of HT29 that resemble goblet cells and produce mucin. However, 12-O-tetradecanoylphorbol-13-acetate and forskolin, which trigger the protein kinase C- and A-dependent signal transduction pathways, respectively, are potent inducers of MUC2 gene expression. 12-O-Tetradecanoylphorbol-13-acetate and forskolin operate through distinct mechanisms, with the former requiring de novo protein synthesis and the latter not. Experiments using specific protein kinase inhibitors suggest that both inducers operate by triggering their respective signal transduction pathways. Nuclear runoff analyses suggest that post-transcriptional (rather than transcriptional) mechanisms are important in the accumulation of MUC2 mRNA. Finally, we show that in several cell lines from human mucinous tumors, characterized by elevated levels of mucin production, MUC2 expression is very high and constitutive compared to forskolin-treated HT29 cells. Thus, the different regulation of MUC2 in HT29 cells and in mucinous tumor cell lines may reflect molecular pathways that characterize colon carcinomas of different histology and pathology.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Modulation of gene expression as a biomarker in colon.

Computer-driven scanning and image processing methodology has demonstrated that genetic inheritance of risk for colorectal cancer in familial polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) families is associated with highly pleiotropic effects on patterns of gene expression in the flat colonic mucosa. The mitochondrial (mt) gene encoding subunit 3 of cytochrome oxidase (COXIII) is one of a panel of cloned sequences which characterize genetic risk. Expression of COXIII decreased in progression of, and risk for, colonic tumors in vivo. Further, metabolizable, unbranched, short-chain fatty acids (SCFAs) elevated expression of mtCOXIII, as well as mtCOXI, in HT29 cells and also elevated mtCOX enzymatic activity. However, expression of nuclear encoded COX subunits were unaffected. These changes may be related to documented alterations in mitochondria structure and function in transformed colonic epithelial cells. SCFAs produced when colonic microflora causes fermentation of fiber are the principle energy source for normal colonic epithelial cells; SCFAs also induce a more differentiated phenotype both in vitro and in vivo. Therefore, a mechanistic link may exist between molecular events in inherited risk and a dietary factor (fiber) which may modulate such risk. In a preliminary intervention trial in collaboration with M. Lipkin, high risk HNPCC patients received daily supplements of 1500 mg CaCO3 per day, which may be protective for development of colorectal tumors. Elevations in COXIII expression were seen in 7 of 12 patients within the first 7 months, followed by complex changes in expression of this sequence.

Adenomatous Polyposis Coli↗
PubMed1991

Effects of fatty acids on expression of genes encoding subunits of cytochrome c oxidase and cytochrome c oxidase activity in HT29 human colonic adenocarcinoma cells.

As the primary and preferred energy source of normal colonic epithelial cells, fatty acids may play a unique role in the differentiation and physiology of these cells. We have shown that expression levels of COXIII, a mitochondrial gene encoding one of the 13 subunits of cytochrome c oxidase, are abnormally low in colon tumors and colonic tissue at genetic risk for developing tumors but increase following in vitro treatment of HT29 human colonic adenocarcinoma cells with the fatty acid butyrate. The present studies investigate the specificity of fatty acids in effecting cytochrome c oxidase subunit expression and enzymatic activity in HT29 cells. The data demonstrate that, depending upon their chain length, metabolizable unbranched fatty acids increase expression of two subunits encoded by mitochondrial genes (I and III) and enhance cytochrome c oxidase activity. However, none of the fatty acids had an effect on expression of two subunits encoded by nuclear genes (IV and Va). These findings suggest that the low levels of COXIII expression exhibited in colonic tumors may represent a limiting factor in the assembly of functional cytochrome c oxidase and contribute to the depressed enzyme activity reported in these tumors. By elevating expression of subunits I and III and enzymatic activity, fatty acids may enhance the potential for cellular respiration. The more differentiated phenotype which is reported in colorectal carcinoma cell lines treated with fatty acids in vitro may be, therefore, associated with correction of metabolic abnormalities in transformed cells.

Adenocarcinoma↗
PubMed1991

Patterns of gene expression that characterize the colonic mucosa in patients at genetic risk for colonic cancer.

We have used a computer-driven scanning and image-processing system to identify a panel of 30 cDNA clones whose pattern of expression in individual biopsy specimens distinguishes the flat, normal-appearing colonic mucosa of patients in two genetic groups at high risk for development of colorectal cancer from that of normal colonic mucosa in low-risk individuals. The two high-risk groups, familial adenomatous polyposis and hereditary nonpolyposis colon cancer, are indistinguishable based on the pattern of expression of the 30 selected clones. This suggests that the extensive pleiotropic effects of the inherited loci, which may play an important role in the mechanism of increased risk and early onset of the disease, are similar in these populations.

Adenomatous Polyposis Coli↗
PubMed1991

Aggressive subtypes of human colorectal tumors frequently exhibit amplification of the c-myc gene.

Expression of the c-myc gene is often elevated in human colorectal tumors, but reported amplification of the locus is rare. Here we demonstrate that modest amplification of c-myc is frequently found in aggressive subtypes of colorectal cancer. Careful quantitation of c-myc copy number has shown amplification in 53.8% (7/13) mucinous tumors, 42.3% (3/7) poorly differentiated tumors and a single poorly differentiated APUD tumor. This contrasts with amplification in 6.9% (2/29) moderately to well differentiated tumors, a value which is in agreement with that in previously published reports. Such changes in gene copy number may represent an important aspect of the genomic alterations which accumulate during the development of colorectal tumors.

Blotting, Southern↗
PubMed1990

Expression of mitochondrial cytochrome c oxidase in human colonic cell differentiation, transformation, and risk for colonic cancer.

In a panel of eight cloned complementary DNA sequences whose level of expression characterize colon cells as transformed in vivo and in vitro, one which may also serve as a marker of risk in familial polyposis and familial colon cancer flat mucosa has been identified as mitochondrial cytochrome c oxidase subunit 3. Mean level of expression of cytochrome c oxidase subunit 3 decreases progressively in colon adenomas and carcinomas relative to normal mucosa in vivo, and returns to higher levels present in biopsies of normal mucosa when the HT29 human colonic adenocarcinoma cell line is induced to differentiate with sodium butyrate. Quantitation of cytochrome c oxidase subunit 3 DNA by dot blots indicated that these changes in expression were not associated with alterations in the number of mitochondrial genomes.

Adenomatous Polyposis Coli↗
PubMed1990

Changes in the number of mitochondrial genomes during human development.

Using a cDNA probe for the mitochondrially encoded third subunit of cytochrome c oxidase (COIII) we found a progressive increase in the number of mitochondrial DNA molecules in specific human tissues during normal fetal development. The data indicate that the tissue, rather than the final number of mitochondrial genomes, apparently plays a dominant role in determining the gestational stage at which the adult complement of this DNA is established.

Blotting, Southern↗
PubMed1990

Absence of detectable deletions in the mitochondrial genome of human colon tumors.

Changes in structure, function, and gene expression of mitochondria have been demonstrated in colorectal tumors (Wong, J.R.; Chen, L.B. Adv. Cell Biol. 2:263-290; 1988). In this report, mitochondrial DNA from 30 primary human colorectal tumors was compared to that of adjacent histologically normal colonic mucosa and human placenta. No evidence for substantial deletions in the major or minor population of mitochondrial DNA was detected, despite the fact that there are extensive deletions throughout the nuclear genome of such tumors. Hence, alterations in mitochondria in colorectal tumors must be dependent upon other mechanisms.

Adenocarcinoma, Mucinous↗
PubMed1987

Expression of cloned sequences in biopsies of human colonic tissue and in colonic carcinoma cells induced to differentiate in vitro.

A computer-based scanning and image-processing system has been developed to quantitate the relative level of expression of each of 4000 cloned complementary DNA sequences in small biopsies routinely removed from the mucosa of normal and neoplastic human large intestine. Individuals have been studied from well-defined population groups in which colonic epithelial cells have progressed to increasingly advanced stages of neoplastic transformation. Comparison of normal colonic mucosa to colonic carcinomas demonstrated alterations in expression of approximately 7% of the cloned sequences; fewer changes were found between benign colonic adenomas and either normal colonic mucosa or carcinomas. A subset of the sequences which change in expression during progression from normal mucosa, to adenoma, to carcinoma showed complementary changes when colon carcinoma cells were induced to differentiate in vitro with sodium butyrate; quantitative correlations between in vivo and in vitro results were highly significant. Comparison of normal colonic mucosa with mucosa from patients with the autosomal dominant disease familial polyposis revealed more extensive alterations in gene expression involving approximately 25% of the clones screened. Flat colonic mucosa in familial polyposis is therefore markedly aberrant and may be highly dedifferentiated, suggesting several possible mechanisms for the very high incidence of cancer that develops in this epithelium.

Adenoma↗
PubMed1987

Overexpression of ras in mucus-secreting human colon carcinoma cells of low tumorigenicity.

We have investigated the expression of the protooncogenes of the myc and ras family in HT29 cells and in three differentiated clonal cell lines derived from this colon carcinoma cell line. In contrast to the decrease in myc expression seen when leukemia cells are induced to differentiate, we have found no changes in expression of the myc gene family in differentiated colon carcinoma cells. However, a greater than 5-fold increase in expression of sequences which hybridize to Ha-ras was observed in cells which secrete mucin, with a smaller increase seen in expression of Ki-ras in the same cells. This increase was not seen in cells which exhibit vectorial transport of water and ions, and which are not mucus-secreting. All differentiated lines were less tumorigenic in nude mice than the parental HT29 cells, irrespective of the level of ras expression. These results are consistent with the reports that ras expression is highest in the most differentiated cells of the colon and is substantially decreased in metastatic human colon tumors as compared to primary colon tumors. The data also suggest that a high level of ras gene expression is a marker for a particular differentiated state in colon cells rather than being directly equated with transformation or tumorigenicity. Hence, the results may reflect on some of the discrepancies concerning ras gene expression in human colon and other tumors which appear in the literature.

Animals↗
PubMed1985

Expression of a mouse long terminal repeat is cell cycle-linked.

The expression of the long terminal repeat (LTR) of intracisternal A particle retroviral sequences which are endogenous to the mouse genome has been shown to be linked to the early G1 phase of the cell cycle in Friend erythroleukemia cells synchronized by density arrest and also in logarithmically growing cells fractionated into cell-cycle compartments by centrifugal elutriation. Regions of homology were found in comparing the LTR sequence to a repetitive Syrian hamster sequence specifically expressed in early G1 in hamster cells.

Animals↗
PubMed1985

Cellular and molecular changes in 3T3 cells transformed spontaneously or by DNA transfection.

Transformed cell lines have been selected following exposure of NIH 3T3 cells to a calcium phosphate precipitate containing DNA from: the human colon carcinoma cell line, SW 480; the cloned Harvey sarcoma virus ras gene; the parental NIH 3T3 cell line; or no DNA (a spontaneous transformant). Unlike the parental 3T3 cells, each of these lines readily formed malignant spindle cell tumors in Swiss nu/mu mice. Southern blots confirmed the presence of the human homologue of the Kirsten ras gene in the cells transformed by SW480 DNA, and the Harvey ras gene in the cells transformed with that cloned sequence. The morphology of each of the lines was different, the cells transformed with the human and viral ras genes being the most aberrant (but not identical) and forming the most extensive foci in culture. These ras transformed lines also exhibited anchorage-independent growth, while the two other transformed lines did not. Both of the ras transformed lines, as well as the spontaneously transformed line, exhibited a pronounced disruption of actin microfilament structure. Two-dimensional gel electrophoresis of 35S-methionine-labeled peptides revealed that these three lines also had a marked decrease in an acidic peptide of 32K daltons.

Actins↗