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B B Knowles

Publications and source records attributed to B B Knowles.

At least 73 records · Page 4Linked to original sources
PubMed1988

Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.

Carcinoma, Hepatocellular↗
PubMed1988

Control of glucose phosphorylation and glucose usage in clonal insulinoma cells.

Glucose metabolism was investigated in two established clonal insulinoma cell lines (RINm5F and HIT) and in a newly developed line of mouse insulinoma cells (IgSV195). The hexokinase capacity in the homogenates of RINm5F cells was 22.1 +/- 3.23 U/g protein, but glucokinase was barely detectable (0.06 +/- 0.013 U/g protein). In contrast, both HIT and IgSV195 cells contained glucokinase (1.5 +/- 0.17 and 1.0 +/- 0.16 U/g protein, respectively) in addition to hexokinase activity. Glucose usage by the intact cells qualitatively reflected the glucose phosphorylation found in the cell-free extracts. RINm5F cells exhibited a high glucose usage rate with one high-affinity component, whereas both HIT and IgSV195 cells showed two components with different glucose affinities. HIT and IgSV195 cells may be useful for a model of pancreatic beta-cell glycolysis.

Adenoma, Islet Cell↗
PubMed1987

Immune control of SV40-induced tumors in mice.

The ability of mice to mount a cytotoxic T-lymphocyte (CTL) immune response to SV40 T-antigen is determined by the H-2 haplotype of the host; H-2b and k mice are high responders and H-2d mice are low responders. Mice of these 3 H-2 haplotypes were challenged with SV40 and their ability to generate and sustain an antibody response to SV40 T-antigen was found to be equivalent. To investigate the role of the different components of the host immune response in controlling growth of SV40-induced tumors, the tumorigenic potential of freshly established cell lines, obtained by SV40 transformation of cells from normal tissues of inbred strains of mice of 6 H-2 haplotypes, was assessed. Each cell line was tumorigenic in athymic and newborn mice but not in adult syngeneic immunocompetent mice. Cells from these initial SV40-transformed lines were then passaged in athymic (nu/nu) mice, re-established in vitro and again transferred into syngeneic animals. Transfer of H-2d SV40 transformants to low or non-responder mice of the H-2d haplotype resulted in tumor formation in some animals. Cells derived from these tumors expressed both the viral encoded T-antigen and the H-2Dd restriction element. Furthermore, the proportion of animals with tumors varied with the strength of their CTL-responsiveness to SV40 T-antigen in association with H-2Dd. Therefore, in H-2d animals, tumor cell growth appears to result from escape of cells from inefficient CTL surveillance. No tumors were formed by transfer of the in vivo selected H-2b or H-2k SV40 transformants to syngeneic high-responder mice. We therefore investigated the role of CTL in the selection of SV40-transformed cells able to escape immune surveillance. Under conditions of stringent immune selection by CTLs, tumorigenic cells that no longer expressed the relevant H-2 class-I restriction element were obtained. Although interaction between the various immune effector mechanisms may play a role in the recognition and elimination of SV40 transformants, our results were consistent with the hypothesis that the SV40-specific CTL response is the predominant control of SV40 tumor growth.

Alleles↗
PubMed1987

Simian virus 40 (SV40)-transgenic mice that develop tumors are specifically tolerant to SV40 T antigen.

The ability to mount an immune response to simian virus 40 (SV40) T antigen was evaluated using mice from two distinct SV40 transgenic lines derived from injection of the same gene construct. Our studies demonstrate functional immune tolerance to SV40 T antigen in a SV40 transgenic line that consistently develops tumors of the choroid plexus by 7 mo of age. Antibodies to SV40 T antigen are undetectable in the serum of these animals; furthermore, mice from this line are unable to generate SV40-specific CTL after primary or secondary immunization with the virus, although they mount a normal CTL response to vaccinia virus when appropriately immunized. In contrast, we find that mice from a second transgenic line of low tumor incidence can mount a humoral response to SV40 T antigen, and upon immunization they generally respond with a vigorous cytotoxic T cell response to SV40 T antigen. These data suggest that specific immune tolerance to the product of an integrated viral oncogene may be induced, and is likely a reflection of the time in development at which the gene product first appears. Immune tolerance or responsiveness to the endogenous oncogene product may in turn play a role in the tumorigenic potential of such genes.

Animals↗
PubMed1987

EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells.

Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.

Cell Line↗
PubMed1986

Red cell antigens P (globoside) and Luke: identification by monoclonal antibodies defining the murine stage-specific embryonic antigens -3 and -4 (SSEA-3 and SSEA-4).

Two globoseries antigens (antigens borne on carbohydrate chains containing globoside), SSEA-3 and SSEA-4, were found on the red cells of the majority of people, but were absent from cells of rare p and Pk individuals which lack globoside. In addition, SSEA-4 was absent from red cells of Luke(-) individuals which nevertheless express the P antigen (globoside) and SSEA-3. The name LKE is proposed for the red cell antigen detected by the Luke serum and by MC813-70, the monoclonal antibody defining SSEA-4. Among the LKE+ individuals, a few showed relatively weak expression of the antigen and were grouped separately as a LKE weak (LKEw) phenotype. Using MC813-70, the frequencies of the 3 phenotypes LKE+, LKEw and LKE- in an English donor population are 0.914, 0.072 and 0.014, respectively.

Animals↗
PubMed1986

Biosynthesis and glycosylation of the epidermal growth factor receptor in human tumor-derived cell lines A431 and Hep 3B.

Biosynthesis of the receptor for epidermal growth factor was investigated in two human tumor-derived cell lines, Hep 3B and A431. When grown in the presence of tunicamycin, both cells expressed a receptor-related species p135, the presumptive aglycosylated form of the biosynthetic precursor, gp145, of the mature form of the receptor, gp165, expressed at the cell surface. Two additional receptor-related species, p115 and p70, were detected when A431, but not Hep 3B, cells were treated with tunicamycin. Furthermore, digestion of the A431 receptor-related proteins with endoglycosidase F resulted in the detection of these three aglycosylated species. P70 appears to be the aglycosylated form of gp95, the presumptive intracellular precursor of the receptor-related species gp120 that is secreted by A431 but not Hep 3B cells; gp120 has a complex pattern of N-linked glycosylation, with consequent molecular weight and charge heterogeneity. P115 may be the aglycosylated form of a third biosynthetic intermediate, possibly a gp135 species detected in the early time points of pulse-chase labeling. Alternatively, p115 and gp135 may be derived co- or post-translationally by Ca2+-mediated proteolysis from p135 and gp145, respectively. The implications of the complexity of the biosynthesis of this molecule with regard to the multiple opportunities it affords the cell to modulate cell proliferation are discussed.

Carcinoma, Hepatocellular↗
PubMed1986

Hepatocellular carcinoma cell line and peripheral blood lymphocytes from the same patient contain common chromosomal alterations.

A cell line derived from the biopsy of a human hepatocellular carcinoma which retains the differentiated phenotype of the liver parenchymal cell is described. Comparison of the integration sites of hepatitis B virus within the cellular genome of the biopsy specimen and within the genome of the multiply-passaged cell line reveals five stable sites of viral integration in the host cell genome. Multiple chromosome abnormalities are found in this cell line, some in the same area of the genome in which abnormalities were found in other human hepatomas. Chromosomes obtained from the peripheral blood cultures of the same patient manifest multiple signs of chromosome instability including breaks, minutes, chromosome pulverization, and acentric fragments, some of which localize to the chromosomal sites involved in the abnormalities in the tumor cell line. Chromosomal instability and/or virus-induced chromosome damage as factors in the etiology of human hepatocellular carcinoma are discussed.

Adult↗
PubMed1986

Immunohistochemical localization of the epidermal growth factor receptor in normal human tissues.

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used to localize this protein in selected normal human tissues. Two patterns of reactivity were recognized: strong linear or granular cell surface staining, and granular cytoplasmic staining. In one tissue, the endometrium, a change in the reaction pattern associated with changes in hormonal stimulation was observed. In some tissues such as epididymis and skin, the antibody showed surface reactivity with cells considered to represent part of the proliferating cell compartment, whereas in liver, pancreas, and prostate, all cells were reactive with the antibody, though the predominant reactivity was localized in the cytoplasm. The differential distribution of the epidermal growth factor receptor to specific cell types and cellular compartments may signify adaptations that permit growth factor responsiveness in a milieu of available ligand.

Adult↗
PubMed1985

Origin of laminin in the extracellular matrix of human tumor xenografts in nude mice.

Monoclonal antibodies reacting exclusively with laminin of human origin and a polyclonal antibody reacting with both murine and human laminin were used to immunohistochemically study the extracellular matrix of four human tumors grown as xenografts in nude mice: a lung carcinoma and a yolk sac carcinoma because they produced cell associated laminin in vitro; and two hepatocellular carcinomas which did not produce cell associated laminin in vitro. The extracellular matrix of the xenografts of the lung carcinoma and the yolk sac carcinoma contained laminin of both human and murine origin. Xenografts of liver carcinoma contained only laminin of mouse origin. This shows that the malignant cells capable of laminin production in vitro contribute this glycoprotein to the extracellular matrix of the solid tumor formed by them in vivo.

Animals↗
PubMed1985

Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development.

Early mouse embryos undergo several morphogenetic processes, such as compaction, trophoblast attachment, and endoderm formation that can be studied in vitro. Several polyspecific and monospecific antisera have been used to perturb these processes in a nontoxic, reversible fashion. One of the antibody-defined molecules, cell CAM 120/80, promotes epithelial cell adhesion, embryo compaction, and endoderm formation. The results suggest the presence of another such molecule(s) involved in these same processes. Evidence is also presented that another set of antibody-defined molecules, GP 140, involved in attachment of somatic cells to the substrate, mediates trophoblast attachment of the mouse blastocyst. The possible role of these molecules in governing the processes leading to cell lineages in the mouse embryo is discussed.

Animals↗
PubMed1985

A human cell-surface antigen defined by a monoclonal antibody and controlled by a gene on human chromosome 1.

An antigen expressed by most human cells, but not erythrocytes, has been defined by a murine monoclonal antibody, TRA-2-10. This antigen is expressed on the surface of human-mouse somatic cell hybrids, and segregation analysis indicates that it is controlled by a gene located on human chromosome 1. From lysates of most human cells, surface-labelled with 125I, TRA-2-10 immunoprecipitates two polypeptides with molecular weights in the range of about 55 000 to 73 000 depending upon the cell line. Since the TRA-2-10 polypeptides from a fibroblast cell strain and a hepatoma cell line from one individual differ, we conclude that the observed difference in molecular weight has an epigenetic origin.

Animals↗
PubMed1985

A fragment of the simian virus 40 early genome can induce tumors in nude mice.

Cell lines transformed by simian virus 40 mutant F8dl (deleted from 0.168 to 0.424 map units, corresponding to the carboxy-terminal 62% of the wild-type simian virus 40 large tumor antigen) are tumorigenic in nude mice. Four of five C3H10T1/2 cell lines transformed by F8dl were tumorigenic in nude mice, whereas two of two wild-type transformants were tumorigenic.

Animals↗
PubMed1985

Chromosomal site of hepatitis B virus (HBV) integration in a human hepatocellular carcinoma-derived cell line.

The single site of integration of hepatitis B virus in the human hepatocellular carcinoma cell line Hep 3B 2-1/7 was found to segregate with human chromosome 12 in somatic cell hybrids. Analysis of metaphase spreads of Hep 3B 2-1/7 following in situ hybridization with pHBV revealed integration at 12q13----q14, a location that coincides with a fragile site, fra (12q13). The possible significance of this location to the development of hepatocellular carcinomas is discussed.

Animals↗
PubMed1985

Characterization of thyroxine-binding globulin secreted by a human hepatoma cell line.

T4-binding globulin (TBG) is a glycoprotein synthesized by the liver and is the principal carrier of T4 and T3 in serum. In this report, we demonstrate that the Hep G2 cell line, derived from a human hepatoblastoma, synthesizes and secretes TBG, the properties of which were characterized. Hep G2 cells secreted TBG into the medium after more than 100 transfers in tissue culture conditions. At confluency and after changing to serum-free culture conditions, TBG accumulation into the medium was linear for 3 days and constituted approximately 0.16% of the proteins synthesized over 24 h. Its abundance relative to albumin is 10-fold greater than that found in normal human serum. TBG secreted by the Hep G2 cells was indistinguishable from native normal human serum TBG, as determined immunologically, by electrophoresis on polyacrylamide gel in denaturing and nondenaturing conditions, and by isoelectric focusing. It also specifically bound T4 and T3, albeit with slightly reduced affinity, and had increased heat lability. Although slightly different from normal serum TBG in caucasians, the physical and biological properties of the Hep G2-derived TBG are similar to those of the variant TBG found in the serum of some healthy Australian Aborigines.

Carcinoma, Hepatocellular↗
PubMed1984

Effects of cis-platinum on embryonal carcinoma cell lines in vitro.

Human and mouse embryonal carcinoma (EC) cells lines were compared with several cell lines of a more differentiated phenotype for their relative sensitivities to the cytotoxic effects of cis-platinum (cis-diamine-dichloroplatinum; CDDP); EC lines were among the most sensitive. Furthermore, several markers of EC cell differentiation were not induced by CDDP, irrespective of the concentration of the drug used. These data provide an explanation for the relative sensitivity of the EC cells in non-seminomatous germ-cell tumors to cis-platinum therapy.

Animals↗
PubMed1984

Biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells.

Using human-specific antibody reagents, we have examined the biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells. Four Mr species (Mr = 70,000, 95,000, 135,000, and 145,000) are detected when cells are subjected to a brief pulse of L-[35S]methionine; an Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel. In pulse-chase experiments, the four lower Mr species appear to bear a precursor relation to the Mr = 165,000 protein. The molecule acquires N-linked oligosaccharide cotranslationally, and two of the species (Mr = 95,000 and 145,000) are susceptible to digestion with endo-beta-N-acetylglucosaminidase H. The Mr = 145,000 and Mr = 165,000 proteins, which become labeled with 125I-epidermal growth factor after treatment of intact cells with a bifunctional cross-linking reagent, are phosphorylated at serine and threonine on identical tryptic peptides.

Carcinoma, Squamous Cell↗