Biomedical subjects

A P Claysmith

Publications and source records attributed to A P Claysmith.

9 recordsLinked to original sources

PubMed1992

Inverse modulation of steady-state messenger RNA levels of two non-integrin laminin-binding proteins in human colon carcinoma.

BACKGROUND: Interactions between cells and the basement membrane glycoprotein laminin are altered during colon cancer progression. Colon carcinoma and normal mucosa cells express a variety of laminin-binding proteins, including the 67-kd laminin receptor (67 LR) and a 31-kd human laminin-binding protein (HLBP31) homologous to the 31-kd human IgE-binding protein/galactoside-binding lectin. PURPOSE: To investigate whether various laminin-binding proteins are differentially expressed in human colon carcinoma, we studied messenger RNA (mRNA) levels of the 67 LR and HLBP31 in matched tumor and adjacent normal mucosa samples from a series of 21 patients. METHODS: Total cellular RNA from tumor and normal mucosa was isolated and analyzed by Northern and slot blot hybridization. In addition, HLBP31 protein levels were assessed by the immunoblot technique. Quantitative laminin affinity chromatography was also used to measure the synthesis of HLBP31 protein in five human cancer cell lines. RESULTS: The steady-state mRNA level of HLBP31 was downregulated (i.e., decreased) in 18 of 21 human colon carcinomas compared with the level in their corresponding normal colonic mucosa. On average, the level of HLBP31 mRNA was decreased 50% +/- 30% (+/- SD) in the colon cancers. The mean ratio of colon cancer HLBP31 mRNA to adjacent normal mucosa HLBP31 mRNA was twofold lower in primary tumors of patients with metastases (0.3 +/- 0.2 SD) than in primary tumors of patients free of metastatic lesions (0.6 +/- 0.2 SD). The differences between the two groups of patients were statistically significant (P less than .05, Wilcoxon-Mann-Whitney test). We have previously shown that the ratio of colon cancer 67 LR mRNA to corresponding normal mucosa 67 LR mRNA was increased in the same patient population. When the two ratios (ratio of cancer to normal HLBP31 mRNA and ratio of cancer to normal 67 LR mRNA) were compared, HLBP31 mRNA/67 LR mRNA was significantly lower (P less than .05) in primary tumors with metastases (mean +/- SD, 0.3 +/- 0.2) than in primary cancers without metastases (mean +/- SD, 0.7 +/- 0.5). The steady-state level of HLBP31 mRNA was directly correlated with the amount of HLBP31 protein in both colon tissue samples and human cancer cell lines. CONCLUSION: HLBP31 mRNA expression in colon cancer tissues is modulated inversely to that of 67 LR mRNA expression. The down-regulation of HLBP31 appears to be associated with the metastatic capabilities of colon cancer cells. IMPLICATIONS: Prospective studies on a large cohort should determine if the systematic detection of HLBP31 and 67 LR protein and/or mRNA can be a valuable adjunct in the prognostic evaluation of primary colon cancers.

Amino Acid Sequence↗

PubMed1992

Detection of laminin receptor mRNA in human cancer cell lines and colorectal tissues by in situ hybridization.

The 67-kd high-affinity laminin receptor (67 LR) is a gene product whose expression appears to be associated with the invasive and metastatic phenotype of a variety of human cancer cells. Northern blot hybridization has been routinely used to quantify the level of 67 LR mRNA from total cellular RNA extracts of homogenized tissue specimens or in vitro grown cell populations. This technique is useful to assess the average expression of the 67 LR mRNA of a particular sample but does not provide information about expression in specific cell types nor about heterogeneity of expression from cell to cell. In this study, we analyzed the expression of 67 LR mRNA in four human cancer cell lines with varying degrees of expression of 67 LR protein (renal cancer A-704, breast carcinoma MCF-7/4 and MCF-7/7, and pancreatic cancer Panc-1) using in situ hybridization performed with 67 LR riboprobes. Total cellular RNA was simultaneously extracted from the cell lines and hybridized on Northern blots with a 67 LR cDNA probe to assess the validity of the mRNA detection by in situ hybridization. Sixty-seven LR mRNA expression was higher in Panc-1 and MCF-7/4 cells than in MCF-7/7 and renal carcinoma A-704. There was a direct correlation (R2 = 0.88) between the in situ hybridization analysis and the mRNA levels detected by Northern blot analysis. The in situ hybridization method showed a heterogeneous expression of the 67 LR mRNA in the four cell lines with different subpopulations of cells showing a range from negative to high levels of the message. Sixteen freshly frozen human colorectal tissues (seven adenocarcinomas, five matched normal mucosae, and four adenomas) were also analyzed by in situ hybridization. The 67 LR mRNA was localized in normal and neoplastic epithelial cells. Adenocarcinoma cells showed a 1.6- to 5-fold higher expression (P < 0.02 according to the Wilcoxon-Mann-Whitney test) than did epithelial colonic cells from normal mucosae or adenomas. The signal tended to be stronger in poorly differentiated carcinomas and carcinomas with metastases than in moderately differentiated and nonmetastatic tumors. We conclude that the high expression of 67 LR mRNA in colorectal tumors is due to an increased production by tumor cells. Furthermore, in situ hybridization is an effective method to detect the expression of LR mRNA in cultured cell lines as well as in frozen tissue sections.

Adenoma↗

PubMed1991

Biosynthesis of the 67 kDa high affinity laminin receptor.

High affinity interactions between cells and laminin are mediated, at least in part, by the 67 kDa laminin receptor (67 LR). A 37 kDa nascent polypeptide (37 LRP), predicted by a full length cDNA clone and obtained by in vitro translation of hybrid-selected laminin receptor mRNA, has been immunologically identified in cancer cell extracts as the putative precursor of the 67 LR. In this study, we used affinity purified antibodies developed against cDNA-deduced 37 LRP synthetic peptides in pulse chase experiments and demonstrated a precursor-product relationship between the 37 LRP and the 67 LR. Immunoblot, pulse chase and immunofluorescence experiments showed that transient transfection of the full length 37 LRP cDNA clone induced a dramatic increase in the synthesis of the 37 LRP but not of the mature 67 LR. We propose that the 67 LR results from the association of two gene products: the 37 LRP and a polypeptide yet to be identified.

Animals↗

PubMed1990

Immunodetection of the metastasis-associated laminin receptor in human breast cancer cells obtained by fine-needle aspiration biopsy.

Fine-needle aspiration biopsy of the breast is a very useful technique for the evaluation of a suspect lesion before surgical removal. Increased expression of the 67-kd laminin receptor has been associated with the metastatic phenotype of cancer cells, particularly in colon and breast cancers. In this study, the expression of laminin receptor was evaluated using the immunoperoxidase technique in 81 breast aspirates (26 benign and 55 neoplastic lesions). Cells obtained from benign samples exhibited a low level of laminin receptor antigen detected by affinity-purified antibody raised against a cDNA-derived laminin receptor peptide. In contrast, 71% of smears obtained from malignant breast lesions contained cells that were strongly stained by the antibody. Heterogeneous expression of the laminin receptor was noted in both breast aspirates and fixed tissue specimens. These data suggest that the immunodetection of laminin receptor in cells obtained by fine-needle aspiration of breast lesions could be a valuable adjunct in the prognostic evaluation of breast lesions.

Antibodies↗

PubMed1989

Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor.

The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues and multiple repeat sequences. The precursor of the laminin receptor is apparently smaller than the 67-kilodalton protein isolated from tissue. The apparent molecular weight on SDS-polyacrylamide gels of the rabbit reticulocyte cell-free translation product of selectively hybridized laminin receptor mRNA is 37,000. Antisera to three different domains of the cDNA-predicted receptor were used to study the relationship between the 37- and 67-kilodalton polypeptides. Antisera to cDNA-deduced synthetic peptides of the receptor immunoprecipitated a 37-kilodalton band both from cell-free translation products and from pulse-labeled cell extracts. On immunoblots of cell extracts, one antisynthetic peptide antiserum recognized only the 67-kilodalton receptor, while another antiserum identified both 37- and 67-kilodalton polypeptides, suggesting a precursor-product relationship between the two polypeptides.

Amino Acid Sequence↗

PubMed1986

Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin.

The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells. This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin.

Amino Acid Sequence↗

PubMed1985

Pachydermodactyly.

Pachydermodactyly is a rare distinct form of fibromatosis characterized by dense fibrosis and fibroblastic proliferation around one or more proximal interphalangeal joints. Pachydermodactyly affects the appositional and dorsal aspects of proximal digits II-V. The lateral aspects of the index fingers, the medial aspect of the fifth digits, and the thumbs are usually unaffected. A typical patient was a young adult man with a long history of idiopathic asymptomatic swelling in the fingers. Skin biopsy specimens obtained from three patients with pachydermodactyly showed marked thickening of the dermis with extension of collagenous fibers into the subcutaneous tissues. There was poor demarcation between the papillary and reticular dermis. The cytology of the fibroblasts appeared benign. Isolation of collagen from the involved sites revealed collagen Types I, III, and V in a pattern typical of fibromatoses; i.e. increased Types III and V. Compared with the collagen profile of normal reticular dermis, increased numbers of fine-diameter collagen fibers were notable in electron micrographs.

Adolescent↗

PubMed1981

Selective localization of wild type and mutant mouse hepatitis virus (JHM strain) antigens in CNS tissue by fluorescence, light and electron microscopy.

Demyelination may be induced by several different pathogenetic mechanisms. We have been utilizing mouse hepatitis virus (MHV) to study virus-induced demyelination in the central nervous system (CNS). To learn whether the different disease phenotypes in 4-week-old mice, caused by wild type (a model for fatal encephalomyelitis) or mutant ts8 (a model for primary demyelination), is due to an altered cellular tropism, we have developed an immunolabeling technique to evaluate critically the localization of MHV antigens in the unique cells of the CNS. Using mouse derived L-cells and primary neuronal cells in vitro, we determined an appropriate fixative (4% paraformaldehyde and 0.5% glutaraldehyde) that both preserved MHV antigenicity and cell structure. These studies in vitro showed the presence of MHV antigens on the surface of cells. Utilizing immunoperoxidase labeling as developed, we studied the localization of MHV antigens in vivo. MHV antigens associated with wild type (wt) virus were localized in neuronal cells as well as oligodendrocytes, which might account for the encephalomyelitis and primary demyelination, respectively. In contrast, MHV antigens associated with ts8 were localized rarely in neurons but commonly in oligodendrocytes. This might account for the uncommon occurrence of fatal encephalomyelitis, but the frequent presence of primary demyelination. Of interest was the finding of viral antigens during MHV infection in the cytoplasmic processes of oligodendrocytes surrounding intact myelin sheaths. We conclude that the different disease phenotypes caused by wt and mutant ts8 reflect differences in the cellular tropism of the two viruses for cells in the CNS.

Animals↗

PubMed1978

Lymphocyte functions and subpopulation distribution in marmosets.

Marmoset lymphocytes were highly reactive to general mitogens, participate in direct cytotoxicity (purportedly mediated by T-cells) and particpate in an antibody dependent lymphocyte cytotoxicity assay (K-cell). The distribution of lymphocyte subpopulations among various species showed that the percent T-cells ranged from 46.4 to 66.6% in different species while complement receptor cells ranged from 10.3 to 19.0%. Surface immunoglobulin (SIg) stable B-cells ranged from 10.6 to 16.4% while the SIg labile L-cells ranged from 26.9 to 37.5%. A fourth receptor, Fc, was demonstrated on 23.7 to 31.7% of the marmoset lymphocytes.

Animals↗