Biomedical subjects
Publications and source records attributed to K Narumi.
We studied the effects of 5 kinds of catechins on the adhesion of mouse lung carcinoma 3LL and melanoma B16F10 cells to the fibronectin substratum. (-)-Epicatechin gallate and (-)-epigallocatechin gallate were active in inhibiting the 3LL cell adhesion, while (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin were inactive. Gallate-containing catechins also impaired adhesion and/or spreading of B16F10 cells.
The accurate diagnosis of mesothelioma remains difficult despite advances of diagnostic technique. And specific monoclonal antibody (McAb) against mesothelioma have not been reported. In an attempt to develop mesothelioma specific McAb(s), spleen cells from a mouse immunized with isolated tumor cells were fused to a drug resistant mouse myeloma cell lines. Over 200 hybridomas were assayed for their preferential reactivity with mesothelioma cell lines or mesothelioma tumor biopsy tissues. Two monoclonal antibodies 2A3 and 4E1 were identified that bound 6/7 of the mesotheliomas, tested, but did not bind to the majority, 11/13 (for 2A3) and 12/13 (for 4E1), of other lung tumor types. Based upon western blot analysis of one and two-dimensional gels and upon the distribution pattern of the antibody recognized molecule in mesotheliomas and non-mesothelioma lung tumors, 2A3 binds to the cell adhesion molecule CD44. While the specificity of 4E1 has not yet been unequivocally established it appears to recognize a variant form of the CD44 molecule.
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Previously we reported that over 75% of human non-small cell lung cancers overexpress the beta 1 integrin VLA-2 on their surface and show an increase in the mRNA encoding the alpha-2 chain of this integrin. These results suggested the possibility that the overproduction and overexpression of one or more of the beta 1 integrin may be involved in the pathogenesis of human lung tumors by modulating the invasive and/or metastatic potential of the tumor. We report here the generation and characterization of multiple clones of tumor cells derived from the primary culture of cells obtained from biopsy tissue of an aggressive human squamous cell lung tumor. We show that these tumor clones (or clonotypes) exhibit seven different yet stable phenotypes with respect to the expression of five members of the beta 1 integrin family. These results illustrate that a primary human lung tumor consists of multiple subpopulations of cells that while indistinguishable by ultrastructure are heterogeneous with respect to their beta 1 integrins. The availability of these distinct tumor clonotypes derived from a single tumor biopsy have made it possible to test the assumption that the beta 1 integrins play a role in tumor progression. The feasibility of this approach is demonstrated here by the intravenous inoculation of different human tumor clonotypes into severe combined immunodeficient (scid) mice. Our preliminary results with a pair of tumor clonotypes differing in VLA-1 and VLA-2 expression level reveal that the clonotype with high level of VLA-1 and VLA-2 displays a substantial increase in the experimental engraftment and metastasis of the human tumor cells in scid mice.
We studied the effects of 5 kinds of catechins on the adhesion of mouse lung carcinoma 3LL cells to the monolayer of bovine lung endothelial cells. (-)-Epicatechin gallate and (-)-epigallocatechin gallate were active in inhibiting the 3LL cell adhesion, while (+)-catechin and (-)-epicatechin were inactive. (-)-Epigallocatechin showed a considerable cytotoxicity. These data suggest that the specific chemical structure is required to exert the inhibitory activity of catechins and the search for the cellular binding protein(s) bound to these inhibitory catechins would provide a clue to clarify the mechanism of interactions between tumor cells and endothelial cells.
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We investigated the expression of laminin in two cell lines with different metastatic potentials established from murine Lewis lung carcinoma. Immunostaining of the cells with anti-laminin antibody and Northern blot analysis of laminin mRNA demonstrated that the high metastatic clone expressed less laminin than the low metastatic one. In contrast, expressions of 67 kDa-laminin receptor were at similar levels between these two lines. These findings show the possibility that endogenous laminin may contribute to the difference in metastatic properties in the murine Lewis lung carcinoma cell lines examined.
Cell-adhesive protein laminin and its specific receptor play an important role in the processes of cancer proliferation, invasion and metastasis. In the present study, we cloned the cDNAs of the 67-kDa laminin receptor both from a human lung cell line (IMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-kDa-laminin receptor mRNA was higher in SBC3 than in IMR90.
Gene expression of the precursor of the 67kDa-laminin receptor was examined by Northern analysis using 11 established human lung cancer cell lines and 25 lung cancer tissues obtained by operation. As a result, one transcript, the size of which was 1.2 kb was shown in all cell lines and tissues examined. An increased level of mRNA was demonstrated in cell lines which proliferated rapidly and in small cell lung cancer cell lines. It was also indicated that gene expression of the laminin receptor was up-regulated especially in small cell lung cancer tissue. In this context, 67kDa-laminin receptor appears to be a marker for biological aggressiveness of human lung cancer.
Complimentary DNA for the 67kDa-laminin receptor was cloned from the cDNA library derived from a human lung cancer cell line, and the nucleotide sequence was determined. Expression of the gene was estimated by Northern analysis in various types of human lung cancer. As a result, increased expression of the laminin receptor was demonstrated especially in the cell types of small cell cancer and bronchioloalveolar cell cancer, which are aggressive biologically. Antibody raised against a partial sequence of the polypeptide was prepared for immunodetection of 67kDa-laminin receptor. It was shown that immunohistochemistry with the antibody could be applied to diagnostic use in lung cancer. The results also suggest that the laminin receptor polypeptide is not necessarily a membrane-associated protein and may function without further processing to the receptor.
Cell-adhesive proteins such as laminin and fibronectin and their specific receptors play an important role in the processes of cancer proliferation, invasion, and metastasis. In the present study, we cloned the cDNA of 67 kDa-laminin receptor both from human lung cultured cell line (IMR90) and from human small cell lung cancer (SBC3), and determined the nucleotide sequences. In addition, the expression of mRNA in 11 lung cancer cell lines with various cell types was estimated by the method of Northern blot hybridization. As a result, 1.2 kb-message was detected in all cancer cell lines examined. It was also demonstrated that the mRNA level of 67 kDa-laminin receptor was inversely proportional to the population doubling time of the cell line (r = -0.80).
We studied the effects of various protein kinase inhibitors on the attachment of mouse lung carcinoma 3LL cells to the fibronectin (FN) substratum. Calmodulin antagonists (W-7 and W-13) and myosin light chain kinase inhibitors (ML-7 and ML-9) exhibited the inhibitory effect for the attachment, while inhibitors of protein kinases A and C were ineffective. Since Arg-Gly-Asp-containing hexapeptide blocked the attachment, cell surface FN receptor appeared to be involved in this mechanism. These results support the hypothesis that the cell attachment requires the rearrangement of the cytoskeleton in association with the phosphorylation of myosin light chain which would lead to the clustering of the cell surface FN receptors.
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1. Mycobacterium phlei (A.T.C.C. 356) cells were incubated with (14)C-labelled short-chain fatty acids and the 6-O-methylglucose-containing lipopolysaccharides that became esterified with radioactive acyl groups were isolated. The pattern of labelling of these lipopolysaccharides with the different acyl groups, the effects of different conditions on labelling patterns, and the kinetics of the turnover of (14)C-labelled acyl groups were studied. 2. The labelling patterns are summarized as follows. [1-(14)C]Acetate was incorporated into all of the acyl groups. [1-(14)C]Propionate led to labelling of propionate and succinate, while [1-(14)C]isobutyrate was incorporated mostly as such, along with a trace amount in iso-octanoate. 3. Under the conditions of the experiments, [1-(14)C]acetate was rapidly incorporated into succinyl (3-carboxypropionyl) and octanoyl groups, whereas the acetyl groups themselves were labelled more slowly. Radioactivity in propionyl and succinyl groups, originating from [1-(14)C]propionate, attained maximum values and then gradually decreased in both. Incorporation of [1-(14)C]isobutyrate proceeded slowly but reached a plateau and remained constant. While n-butyrate is not a normal constituent of methyl-glucose-containing lipopolysaccharides, it was incorporated as such when n-[1-(14)C]-butyrate was supplied in the medium. 4. [1-(14)C]Acetyl groups were readily displaced by unlabelled acetate. On the other hand, the specific radioactivity of the succinyl group continued to increase during a 3h incubation with unlabelled succinate. Propionyl and succinyl groups, labelled by [1-(14)C]propionate, were displaced slowly by unlabelled propionate or succcinate. The isobutyryl group of the lipopolysaccharides did not turn over, in contrast to the results obtained with the other acyl substituents.
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