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M L Frazier

Publications and source records attributed to M L Frazier.

At least 37 records · Page 2Linked to original sources
PubMed1998

Loss of p21WAF1/Cip1 protein expression accompanies progression of sporadic colorectal neoplasms but not hereditary nonpolyposis colorectal cancers.

p21 (p21WAF1/Cip1), a cyclin-dependent kinase inhibitor, induces G1 arrest and can inhibit the activity of the proliferating cell nuclear antigen (PCNA). We analyzed p21 expression during colorectal tumorigenesis, its association with its transcriptional regulator p53, and its relationship to rates of cell proliferation and apoptosis. p21 and p53 protein expression were examined in sporadic tumors and hereditary nonpolyposis colorectal cancers (HNPCCs) by immunohistochemistry (IHC) and immunoblotting. Apoptosis was examined using a DNA nick end-labeling assay, and cell proliferation was examined by PCNA staining. In normal colorectal epithelia, nuclear p21 staining was uniformly detected in crypt cells of the superficial compartment (upper one-third) that stained negatively for PCNA. p21 and PCNA expression were, therefore, mutually exclusive. In sporadic cases, a decrease in the frequency of p21 expression accompanied adenoma development and progression to carcinoma. Specifically, p21 was detected in 12 of 16 (75%) adenomas and 10 of 32 (31%) carcinomas. In contrast to sporadic cases, HNPCCs with known mutations in DNA mismatch repair genes expressed p21 in 12 of 15 (80%) carcinomas. An inverse relationship between p21 and p53 was observed wherein mutant p53 proteins were detected in 4 of 15 (27%) HNPCCs versus 22 of 32 (69%) sporadic carcinomas. Although p21+ carcinoma cells were generally negative for p53, IHC revealed that some carcinoma cells expressed both p21 and p53 proteins. Furthermore, p53-mutated SW480 colon carcinoma cells were found to coexpress p21 and p53, suggesting that p21 can also be activated by a p53-independent mechanism. No association was found between p21 or PCNA and apoptotic labeling indices in adenomas or carcinomas. In conclusion, a decrease in p21 expression accompanies neoplastic progression in sporadic cases but not in HNPCCs. This finding appears related to p53 status in that the frequency of p53 expression was significantly reduced in HNPCCs compared to sporadic cases, suggesting a difference in their molecular pathways of tumorigenesis.

Colorectal Neoplasms↗
PubMed1998

Few point mutations in elongation factor-1gamma gene in gastrointestinal carcinoma.

Elongation factor-1 (EF-1) gamma is overexpressed in a high proportion of gastrointestinal cancers. The mechanism of overexpression has not been determined. The purpose of this study was to examine cDNA specimens from pancreatic and colorectal cancer for mutation in this gene, which would allow us to determine whether gene mutations are responsible for overexpression of EF-1gamma. In one colorectal carcinoma, we detected an A-->G transition at amino-acid codon 158 (T-->C in the sense strand) resulting in a change from a leucine to a serine. The base change was not detected in cDNA isolated from normal-appearing tissue from the same patient. We did not find mutations in another five colorectal carcinoma and five pancreatic cancer samples. Thus, although we detected a mutation in one tumor, the frequency of mutations was too low to account for the high frequency of overexpression of the EF-1gamma RNA in colorectal cancer. We also investigated other possible mechanisms of overexpression of the EF-1gamma RNA in this study. Slot-blot analysis of DNA isolated from colorectal cancers showed that the overexpression was not due to gene amplification. Using serum starvation to synchronize cultured cells, we showed that the overexpression was also not due to an increase in the number of cycling cells, as occurs in cancer. Using Southern blot analysis, we were unable to detect genome rearrangements that could have been responsible for the overexpression. In conclusion, the mechanism for overexpression of the EF-1gamma gene in colorectal and pancreatic cancer remains unknown. However, mutations in the coding sequence of the gene, gene amplification, and gene rearrangement do not account for the high frequency of overexpression of this gene, and the overexpression is not due to an increase in the number of cycling cells.

3T3 Cells↗
PubMed1997

Fine mapping of a genetic locus for Peutz-Jeghers syndrome on chromosome 19p.

Peutz-Jeghers syndrome (PJS) was recently mapped in a single report to the telomeric region of chromosome 19p (A. Hemminki et al., Nat. Genet., 15: 87-90, 1997). Our studies confirm this location and provide further localization of the PJS locus. In the five families examined, there were no recombinants with the marker D19S886. The multipoint log odds score at D19S886 is 7.52, and we found no evidence for genetic heterogeneity. We also found that all carriers expressed the PJS phenotype and no noncarriers displayed PJS sequellae, indicating complete penetrance with no sporadic cases.

Chromosome Mapping↗
PubMed1997

Cis-acting elements required for expression of the nonspecific cross-reacting antigen gene in colorectal carcinoma.

BACKGROUND & AIMS: The nonspecific cross-reacting antigen (NCA) is a cell adhesion molecule, and the messenger RNA for NCA is overexpressed in 92% of colorectal carcinomas. The aim of this study was to determine the cis-acting elements that may be responsible for the expression of NCA. METHODS: Deletion mutants of the 5' flanking sequence and first intron were ligated into chloramphenicol acetyltransferase expression vectors, transfected into Chinese hamster ovary (CHO), DiFi, and HT-29 human colorectal carcinoma cells, and BxPC-3 and MDAPanc-28 human pancreatic carcinoma cells. The amount of acetylated chloramphenicol was determined to show the presence and activity of cis-acting sequences. RESULTS: The 5' flanking sequence functions as a promoter in all of cell lines and contains negative regulatory and enhancer sequences. The minimal promoter is active in Chinese hamster ovary and HT-29, though not in MDAPanc-28 cells. The first intron contains a silencer capable of suppressing a heterologous promoter. CONCLUSIONS: The results show cis-acting sequences within and 5' to the NCA gene, which appear to play a role in the expression of this gene in malignant tissues. Some of these sequences function in a cell type-specific manner. Further studies of these elements may provide insight into the mechanisms of the abnormal growth patterns of malignant cells.

Animals↗
PubMed1996

Mutation of the hMSH2 gene in two families with hereditary nonpolyposis colorectal cancer.

We examined 18 unrelated individuals who have colorectal cancer or cancers associated with the HNPCC syndrome and have a family history of cancer for mutations in exon 13 of the hMSH2 gene. Two of the 18 individuals had the same previously unreported single-base deletion in codon 705 of hMSH2, resulting in a frame-shift mutation. Two other individuals had a T-to-C base change in the intron sequence at -6 position of the splice acceptor site at the 5' end of exon 13 which has previously been reported to be a polymorphism found in normal individuals (Leach et al., 1993). Nucleotide sequence changes were not detected in the remaining 14 individuals. We examined DNA from additional family members of the two subjects with codon 705 mutations. Of these, two individuals were identified with the mutation who were older than 50 years and who are apparently cancer free. The occurrence of breast cancer in both families (including one individual with a confirmed codon 705 mutation) suggests that breast cancer may be a part of the HNPCC syndrome. This is the first study to describe mutations in the hMSH2 gene in families that do not fit the definition of HNPCC. Because both families with the mutations at codon 705 failed to meet the Amsterdam criteria for HNPCC (Vasen et al., Dis Colon Rectum 34:424-425, 1991), our findings suggest that these criteria should now be reconsidered for purposes of diagnosis of HNPCC.

Adult↗
PubMed1996

Molecular diagnosis of exocrine pancreatic cancer using a percutaneous technique.

BACKGROUND: The K-ras oncogene is activated by point mutations at codon 12 in most patients with exocrine pancreatic cancer. Mutant-enriched polymerase chain reaction (PCR) amplification can enhance the detection of mutated K-ras. This technique was applied to patients undergoing percutaneous fine-needle aspiration (FNA) biopsy of suspect pancreatic lesions. METHODS: Twenty-five patients underwent percutaneous FNA of the pancreas for cytologic and molecular analysis. After preparing cytologic smears, the 22-gauge needle and syringe used for FNA were rinsed in RPMI-1640. The specimen was centrifuged, and DNA was extracted from the supernatant and subjected to mutant-enriched PCR using appropriate mismatched primers that introduce a BstNI restriction endonuclease clevage site at codon 12 of wild-type, but not mutant, K-ras. After digestion with BstNI, the DNA was reamplified. To increase assay sensitivity, the final five PCR cycles were completed incorporating 5 microCi of (alpha-32P)dCTP. The DNA was then redigested and subjected to gel electrophoresis and autoradiography. RESULTS: The median amount of DNA retrieved per specimen was 3.33 micrograms. Mutant K-ras was detected as a band of 143 bps; residual wild-type DNA was seen as a 114-bp fragment. Twenty-one of 25 specimens demonstrated mutated K-ras DNA. Two patients with nondiagnostic cytology results had mutated K-ras DNA; adenocarcinoma of pancreatic origin was confirmed in both cases after pancreatectomy. CONCLUSION: The molecular diagnosis of pancreatic cancer through identifications of mutations in K-ras can be readily performed on specimens obtained by percutaneous FNA. As aggressive multimodality management of this disease becomes more common, pretreatment analysis of molecular determinants may have greater clinical significance.

Adenocarcinoma↗
PubMed1996

Pancreatic adenocarcinoma cell line, MDAPanc-28, with features of both acinar and ductal cells.

CONCLUSION: We established a new human pancreatic adenocarcinoma cell line, MDAPanc-28. Studies on this new line indicate that it expressed both acinar and ductal gene products suggesting that the patterns of gene expression in the pancreatic adenocarcinoma from which this cell line arose have features similar to those of the protodifferentiated cells hypothesized by Rutter and his colleagues for the developing pancreas (1,2). BACKGROUND: The cell line arose from a tumor that, like most pancreatic adenocarcinomas, was ductal on the basis of its histological appearance. METHODS: Once the cell line was established in culture, they were subjected to cytogenetic analysis and tested for their ability to grow in nude mice. RNA from the cells was analyzed by Northern blot analysis and PCR of reverse transcribed cDNA for the expression of both acinar and duct cell gene products. DNA was analyzed for the presence of mutated K-ras at codon 12. RESULTS: The cell line expressed trypsin and ribonuclease RNA, which are considered to be acinar cell markers, and carbonic anhydrase II (CAII), which is considered to be a duct-cell markers. The histological appearance of xenografts in nude mice was similar to that of the tumor from which the cell line was established. The chromosome number varied between 46 and 60.

Adenocarcinoma↗
PubMed1996

Evidence for a major gene for type II diabetes and linkage analyses with selected candidate genes in Mexican-Americans.

We have carried out two independent family studies in low-income Mexican-Americans from San Antonio, Texas. In the first study, probands were ascertained at random without regard to any medical condition (658 examined individuals from 50 families), and in the second study, probands were subjects with type II diabetes identified in a prior epidemiological survey (523 examined individuals from 29 families). Pedigrees ranging in size from 2 to 45 family members (median 11) in the first study and from 2 to 50 family members (median 12) in the second study were examined. Diabetes was diagnosed according to World Health Organization criteria. In both sets of families, segregation analyses revealed support for a major gene with an autosomal dominant mode of inheritance influencing early age of onset of diabetes. Non-Mendelian inheritance was rejected in both data sets. Individuals with the early age of onset allele had a mean age of diabetes onset of 51 years in the first data set and 60 years in the second data set. In the first data set, the major gene accounted for approximately 70% of the phenotypic variance in age of onset of diabetes, and there were no residual family effects once the major gene effect was taken into account. In the second data set, the major gene accounted for approximately 50% of the phenotypic variance, and residual family effects were statistically significant. Linkage analyses were performed with 11 candidate genes, and tight linkage with diabetes was rejected for Rh blood group, glucose transporter 2, fatty acid-binding protein, tumor necrosis factor beta, glucokinase, and lipoprotein lipase. A logarithm of odds (LOD) score of 0.92 at a recombination fraction of 0.05 was observed for insulin receptor substrate 1. This LOD score corresponds to a chi2 of 4.24 (P = 0.039).

Alleles↗
PubMed1995

Evidence for linkage of postchallenge insulin levels with intestinal fatty acid-binding protein (FABP2) in Mexican-Americans.

Single genes with large effects may contribute to insulin resistance or influence susceptibility to non-insulin-dependent diabetes mellitus (NIDDM). In the Pima Indians, results from sib-pair analysis have suggested that a gene on chromosome 4q influences both fasting insulin levels and maximal insulin action. We conducted sib-pair and logarithm of odds (LOD)-score linkage analysis to seek evidence for linkage between genes influencing insulin levels and chromosome 4q loci. Analyses were conducted on nondiabetic individuals from 28 different families participating in the San Antonio Family Diabetes Study. All subjects received a 2-h oral glucose tolerance test. Fasting insulin levels were measured in 382 nondiabetic individuals, and 2-h insulin levels were measured in 366 individuals. Initial sib-pair linkage analysis revealed a possible association between 2-h post-glucose challenge insulin levels and the intestinal fatty acid-binding protein (FABP2) locus located in the region of chromosome 4q28-31 (P = 0.006). Subsequent sib-pair linkage analysis of 11 additional chromosome 4q markers supported this hypothesis. We next conducted segregation analyses to estimate allele frequencies and other model parameters for the putative locus influencing 2-h insulin levels. Results of LOD-score linkage analysis indicated possible linkage between the major gene described by the segregation model and FABP2. Using combined segregation and linkage analysis, we obtained a LOD-score of 2.80 at recombination frequency of 0.0 between FABP2 and the putative locus influencing 2-h insulin levels. The maximum likelihood estimate of the allele associated with low insulin levels was 0.21.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier Proteins↗
PubMed1994

Expression of acinar and ductal products in Capan-1 cells growing in synthetic serum and serum-free media.

BACKGROUND: Capan-1 is a human pancreatic adenocarcinoma cell line of presumed ductal origin. This is based on the histologic appearance of the tumor from which it arose. Yet considerable controversy exists regarding the actual cell of origin for these exocrine carcinomas. Two acinar antigens, ribonuclease and trypsin, were analyzed in cells growing in synthetic serum. METHODS: Capan-1 cells were adapted to grow in basal medium supplemented with synthetic serum, because fetal bovine serum (FBS) normally used to culture cells contains bovine ribonuclease, which can interfere with measurements of the ribonuclease secretion. These cells were also adapted to grow in different serum-free media, allowing us to determine its minimal growth requirements. The presence of ribonuclease in Capan-1 and PANC-1 conditioned media was monitored by activity. Other acinar and ductal markers were monitored using Northern blot analysis. RESULTS: Capan-1, PANC-1, IBF-CP3, and MDAAmp-7 cell lines were successfully adapted to grow in synthetic serum by means of the adaptation protocol reported here. The adaptation of Capan-1 to serum-free media showed that the cells are capable of growing in a medium containing insulin, transferrin, selenium, a nonprotein carrier, and lipoic and linoleic acids. Northern blot analysis showed the expression of carbonic anhydrase II, cytokeratin 18, ribonuclease, and trypsin in Capan-1 cells growing in FBS and synthetic serum. No changes in morphology, karyotype, or gene expression were observed in these cells as a result of the adaptation process. CONCLUSION: The cell line Capan-1 is expressing some ductal as well as acinar products despite its supposed ductal origin. The expression of trypsin at the mRNA level and ribonuclease at mRNA and protein levels is shown in Capan-1 cells. The protein expression will be further investigated as the cell line has been adapted to grow in synthetic serum and serum-free media with no apparent changes with respect to their growth in FBS.

Adaptation, Physiological↗
PubMed1994

Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures.

Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.

Amylases↗
PubMed1993

Regulation of sialomucin production in colon carcinoma cells.

In this study, we investigated the biochemical basis for the induction of sialomucin production in HT-29 LMM human colon carcinoma cells. Previous studies showed that conditioned medium from organ cultures of normal human colonic connective tissues (NCCM) stimulated the biosynthesis of high molecular weight sialoglycoproteins (M(r) 740,000 and 450,000) by colon carcinoma cells in vitro. Using monoclonal antibodies specific for human milk mucin, we determined that the polypeptide core of these sialomucins was the polymorphic epithelial mucin core peptide coded by the MUC1 gene. Northern analysis showed that cells treated with NCCM expressed a higher level of MUC1 poly(A)+ mRNA than untreated cells. The UDP-GalNAC: polypeptide N-acetylgalactosaminyltransferase activity in microsomal fractions from these cells did not change upon NCCM treatment. The ratios of oligosaccharides with various negative charges secreted by HT-29 LMM cells in the presence of benzyl-alpha-D-N-acetylgalactosaminide were not affected by NCCM treatment indicating that the degree of sialylation of O-linked carbohydrate chains is not influenced by NCCM treatment. [3H]Glucosamine pulse-chase labeling studies using antibodies specific for a COOH-terminal unglycosylated region of the MUC1 mucin core peptides suggested that NCCM treatment of HT-29 LMM cells did not change the rate of MUC1-related sialomucin processing.

Blotting, Northern↗
PubMed1993

Overexpression of an elongation factor-1 gamma-hybridizing RNA in colorectal adenomas.

While it is apparent that colorectal carcinogenesis results from a series of genetic alterations manifested phenotypically by the adenoma-to-carcinoma sequence, the early events that occur in the process of tumorigenesis have not been elucidated. We previously demonstrated that human elongation factor-1 (EF-1) gamma-hybridizing RNA was overexpressed in 25 of 29 colorectal carcinomas. To determine if the overexpression of this mRNA occurs early in tumor development, we examined 25 adenomas and corresponding normal-appearing distant mucosae from 20 patients without familial adenomatous polyposis (FAP). We observed overexpression at a level of twofold or more in 14 (56%) of the 25 adenomas, indicating that overexpression of EF-1 gamma RNA is often a relatively early event in the development of non-FAP colorectal cancer.

Adenoma↗
PubMed1993

Cytotoxic T-cell clones isolated from ovarian tumour infiltrating lymphocytes recognize common determinants on non-ovarian tumour clones.

CTL-TIL lines have been developed from tumour infiltrating lymphocytes (TIL) from the ascites of patients with ovarian carcinoma, and used to investigate whether common tumour antigens are expressed on allogeneic ovarian tumours epithelial tumour lines derived from colon and pancreatic carcinoma. Three CTL lines expressed preferential cytolytic activity against autologous tumour cells and against certain allogeneic ovarian tumour cells that shared HLA-A2 molecules. Analysis of the target specificity of these CTL lines indicated that they also lysed human colon and pancreatic tumour lines sharing HLA-A2. CTL-TIL clones isolated from these lines were found to lyse HLA-A2+ ovarian, colon and pancreatic tumours, and to recognize clonally distributed common epitopes on pancreas and colon tumour clones. These results indicate that shared tumour antigens can be found among tumours of common epithelial cell origin. These results indicate a novel class of T-cell-definable tumour antigens recognized by tumour-reactive CTL on human tumours and may be significant for understanding of cellular immunity in ovarian cancer, identification of CTL-defined tumour antigens and future adoptive specific immunotherapeutic approaches in ovarian cancer.

Antigens, Neoplasm↗
PubMed1992

Expression of an elongation factor 1 gamma-related sequence in adenocarcinomas of the colon.

The authors have previously reported that a messenger RNA (mRNA) bearing 60% homology to elongation factor 1 gamma in Artemia salina was overexpressed in 7 of 9 pancreatic tumors relative to normal appearing adjacent tissue. The purpose of the present study was to determine if this pattern of overexpression is also detected in colorectal carcinoma. Overexpression was observed in 25 of 29 colorectal carcinomas, relative to normal adjacent tissue. Of them, mRNA was overexpressed in 2 tumors classified as Dukes' D, in 8 of 11 tumors classified as Dukes' B2, and in 15 of 16 tumors classified as Dukes' C2.

Adenocarcinoma↗
PubMed1992

Expression of elongation factor-1 gamma-related sequence in human pancreatic cancer.

A cDNA clone designated pPDC-1 was isolated from a cDNA library prepared against poly(A+)RNA isolated from the human pancreatic adenocarcinoma cell line, Capan-2. The cDNA corresponds to a 1.7-kilobase mRNA that is expressed at higher levels in seven of nine pancreatic tumors than in their corresponding normal tissues. It is also expressed in normal human kidney, intestine, pancreas, stomach, placenta, lung, brain, spleen, and liver. A computer search of the Intelligenetics System of all available nucleotide sequences revealed a 60% homology between the nucleotide sequence of the pPDC-1 cDNA and that of elongation factor-1 gamma from Artemia. The deduced amino acid sequence shared 53% identity with the amino acid sequence for the Artemia elongation factor-1 gamma.

Aged↗