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B B Knowles

Publications and source records attributed to B B Knowles.

At least 55 records · Page 3Linked to original sources
PubMed1992

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor.

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.

Carcinoma, Hepatocellular↗
PubMed1991

Chromosome abnormalities in peripheral blood cells of hepatitis B virus chronic carriers.

Cytogenetic analysis of metaphase chromosome spreads from peripheral blood cells of hepatitis B virus (HBV) chronic carriers and HBV-negative individuals of the same ethnic origin revealed a significantly higher incidence of chromosome breaks and other mitotic aberrations in the HBV chronic carriers. The highest incidence of chromosome breaks was found in chronic carriers who evidenced circulating HBV. Such an association between HBV and these genetic lesions assumes importance in light of the known correlation between HBV chronic carrier status and the high risk of hepatocellular carcinogenesis, where a mutagenic effect of HBV cannot be excluded.

Carcinoma, Hepatocellular↗
PubMed1991

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice.

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.

Adenoviridae↗
PubMed1991

Frequency of SV40-specific cytotoxic T-lymphocyte precursors in two SV40 T-antigen transgenic mouse lines.

Cytotoxic T-lymphocyte precursors (CTL-P) from control C57BL/6 mice and from mice of two simian virus 40 (SV40) T-antigen transgenic lines, 427 and 419, specifically nonresponsive and responsive, respectively, to SV40 T antigen, were quantitated by limiting dilution (LD) after immunization with SV40. CTL-P frequencies for the SV40 T antigen-responsive 419 line transgenic mice were within the range established in C57BL/6 mice, whereas no CTL-P could be demonstrated for the SV40 T antigen-tolerant 427 line mice. These results suggest that deletion or anergy of SV40 T antigen-responsive clones underlies the specific profound tolerance of 427 line mice.

Animals↗
PubMed1991

Abnormalities of chromosome 1 and loss of heterozygosity on 1p in primary hepatomas.

Cytogenetic analysis of eight human hepatoma-derived cell lines and one primary hepatocellular carcinoma biopsy revealed multiple chromosome abnormalities; however, only chromosome 1 was consistently affected by rearrangements. Pseudopolysomy 1 as well as chromosome 1 deletions and/or translocations that resulted in loss of the distal 1p region from at least one copy of chromosome 1 were observed in all but one of the cell lines analysed. Molecular analyses of tumor-derived and normal genomic DNA from six cases of hepatocellular carcinoma and from two of hepatoblastoma, using a panel of chromosome 1p-specific DNA probes indicated allelic loss in the distal 1p region in five of the six hepatocellular carcinomas but not in either hepatoblastoma. These results suggest the location of a gene in the distal 1p region whose functional loss may be involved in hepatocellular carcinogenesis.

Carcinoma, Hepatocellular↗
PubMed1990

EGF receptor down-regulation attenuates ligand-induced second messenger formation.

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.

Angiotensin II↗
PubMed1990

Chromosomal rearrangements in a primary hepatocellular carcinoma.

Chromosome analysis of cells obtained at biopsy from a 65-year-old man with primary hepatocellular carcinoma revealed characteristic abnormalities of chromosomes 1, 5, 6, 9, 13, 16, and 22 in each cell and maintenance of a pseudodiploid chromosome number (46,XY). Five of the chromosomal sites involved in these rearrangements are either in fragile site regions or in regions containing genes that encode cellular oncogenes. Some of the tumor cells manifest mitotic deviations in the form of asynchronies, spiralization, premature centromere division, and non-sister chromatid associations. The significance of these findings to hepatocellular carcinogenesis is discussed.

Aged↗
PubMed1990

Osteosarcomas in transgenic mice expressing an alpha-amylase-SV40 T-antigen hybrid gene.

Mice of a transgenic mouse lineage 501, produced by zygotic injection of the parotid alpha-amylase promoter-SV40 T-antigen hybrid gene, developed osteosarcomas at about 15 months of age. The tumors predominantly involved the axial skeleton, were sometimes multiple, and metastasized to the liver. A cell line derived from a primary tumor produced osteosarcomas on transfer to nude mice. The 501 transgenic lineage thus provides a valuable new model for studying the histogenesis of osteosarcomas.

Animals↗
PubMed1989

Metastatic hibernomas in transgenic mice expressing an alpha-amylase-SV40 T antigen hybrid gene.

Mice transgenic for a hybrid gene containing the liver promoter of the mouse amylase gene (Amy-1a) fused to the SV40 tumor antigen coding region unexpected developed malignant brown adipose tissue tumors (malignant hibernomas). Expression of the alpha-amylase gene had previously been thought to be confined to the liver parotid, and pancreas; however, analysis of white and brown adipose tissue from nontransgenic mice revealed expression of the endogenous Amy-1a gene in these tissues. Gene constructs driven by the Amy-1a liver promoter thus provide a means of targeting gene expression to the adipocyte cell lineage in transgenic mice. Moreover the high frequency of metastases in the liver, lungs, spleen, heart, and adrenals of these mice provides an experimental system in which to study the development of disseminated malignancy.

Adipose Tissue↗
PubMed1989

SV40 T antigen acts as a minor histocompatibility antigen of SV40 T antigen tolerant transgenic mice.

The ability of normal mice to mount an SV40 T antigen-specific cytolytic T lymphocytes response when immunized in vivo with splenocytes from the SV40 T antigen transgenic 427-line mice and restimulated in vitro with SV40-transformed fibroblasts, or when immunized with SV40 and restimulated with 427-line splenocytes, was analyzed. Both immunization schemes resulted in an SV40 T antigen-specific immune response, indicating the presence of SV40 T antigen-positive cells in the spleens of these transgenic mice. Normal mice engrafted with skin from 427 donors showed no rejection of the graft. Thus, SV40 T antigen in transgenic 427-line mice is expressed on an undefined cell type in the spleen and acts as a tissue-specific minor histocompatibility antigen.

Animals↗
PubMed1989

Functional pancreatic beta-cell line from SV40 T-antigen transgenic mouse.

Cell line IgSV195, derived from a pancreatic tumor that arose in an SV40 T-antigen transgenic mouse, retains certain morphological and physiological characteristics of pancreatic beta-cells throughout in vitro and in vivo passage. Insulin secretion is stimulated by exposure of these cells to fetal bovine serum and a combination of 3-isobutyl-1-methylxanthine and glutamine but not by concentrations of glucose in the physiological range. Insulin processing appears to be intact. Neither class I nor class II major histocompatibility complex (MHC) antigens are routinely expressed at the cell surface; however, MHC class I--but not class II--encoded gene products are detected after treatment with recombinant interferon-gamma (IFN-gamma) alone or in combination with tumor necrosis factor. Cytolysis of IgSV195 cells by SV40 T-antigen-specific H-2b-restricted lymphocytes is similarly dependent on IFN-gamma pretreatment. These results emphasize that SV40 T-antigen transgenic mice are likely sources of cell lines that retain their differentiated function in vitro. The IgSV195 cell line provides an accessible model in which to investigate the control of gene expression and function of pancreatic beta-cells.

Animals↗
PubMed1989

Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2.

A total of six established human hepatoma-derived cell lines, including Hep3B, NPLC/PRF/5 (NPLC), Tong/HCC, Hep 10, huH1, and huH2, were screened for their ability to accumulate significant quantities of lipoproteins in serum-free medium. Only two cell lines, Hep3B and NPLC, secreted quantitatively significant amounts of lipoproteins. In a 24-h period the accumulated mass of apolipoproteins (apo) A-I, A-II, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 micrograms/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-II, B, and E was 0.45, 0.05, 0.32, and 0.68 micrograms/mg cell protein per 24 h, respectively. Twenty four-hour serum-free medium of Hep3B cells contained lipoproteins corresponding to the three major density classes of plasma; percent protein distribution among the lipoprotein classes was 4%, 41%, and 56% for very low density lipoprotein ("VLDL"), low density lipoprotein ("LDL"), and high density lipoprotein ("HDL"), respectively. NPLC was unusual since most of the lipoprotein mass was in the d 1.063-1.235 g/ml range. Hep3B "LDL", compared with plasma LDL, contained elevated triglyceride, phospholipid, and free cholesterol. Nondenaturing gradient gel electrophoresis revealed that Hep3B "LDL" possessed a major component at 25.5 nm and a minor one at 18.3 nm. Immunoblots showed that the former contained only apoB while the latter possessed only apoE. Like plasma VLDL, Hep3B "VLDL" particles (30.5 nm diameter) isolated from serum-free medium contained apoB, apoC, and apoE. "HDL" harvested from Hep3B and NPLC medium were enriched in phospholipid and free cholesterol and poor cholesteryl ester which is similar to the composition of HepG2 "HDL." "HDL" from Hep3B and NPLC culture medium on gradient gel electrophoresis had peaks at 7.5, 10, and 11.9 nm which were comparable to major components found in HepG2 cell medium. Hep3B cells, in addition, possessed a particle that banded at 8.2 nm which appeared to be an apoA-II without apoA-I particle by Western blot analysis. The cell line also produced a subpopulation of larger-sized "HDL" not found in HepG2 medium. NPLC "HDL" had a distinct peak at 8.3 nm which by Western blot was an apoE-only particle. Electron microscopy revealed that "HDL" harvested from Hep3B and NPLC medium consisted of discoidal and small, spherical particles like those of HepG2. The "HDL" apolipoprotein content of each cell line was distinct from that of HepG2. ApoA-II at 35% of apolipoprotein distinguishes Hep3B "HDL" from HepG2, which contains only 10%.(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoprotein A-I↗
PubMed1988

High affinity human IFN-gamma-binding capacity is encoded by a single receptor gene located in proximity to c-ros on human chromosome region 6q16 to 6q22.

We have used human-rodent somatic cell hybrids to investigate the regional localization of the IFN-gamma R gene on human chromosome 6 and studied functional and antigenic characteristics of the expressed IFN-gamma R by Scatchard analyses of 125I-IFN-gamma binding and binding of an anti-receptor mAb (A6C5). The data obtained revealed coordinate expression of IFN-gamma- and A6C5-binding capacity as well as competition in binding to chromosome 6-positive hybrids and normal cells, indicating that the A6C5-defined protein is by itself capable of high affinity IFN-gamma binding and, thus, is likely to constitute the major IFN-gamma R protein of distinct cell types. The receptor gene could be allocated to region 6q16 to 6q22, which also contains the c-ros oncogene. Genetic linkage of the IFN-gamma R gene to an oncogene located in a region of non-random chromosomal aberrations may have a causal relationship to the deregulated IFN-gamma R expression in several malignancies.

Antibodies, Monoclonal↗
PubMed1988

Epidermal growth factor-induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma-derived cell line.

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.

Calcium↗
PubMed1988

SV40 T-antigen is a histocompatibility antigen of SV40-transgenic mice.

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigens by the strict criteria that such antigens stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.

Animals↗
PubMed1988

A simple technique to distinguish rat from mouse chromosomes in T cell hybridomas.

Antigen-specific rat mouse T-cell hybridomas, prepared by fusing the BW5147 AKR thynoma with helper T cells from rats immunized with chicken ovalbumin, express both the rat and mouse major histocompatibility complex gene products. Using these functionally defined T-cell hybridomas, we have devised a technique based on the differential staining of the heterochromatin region of the rat and mouse chromosomes to distinguish the species of origin of the chromosomes. This technique should prove useful for screening potential hybrid cell lines and should aid in differentiating mouse from rat chromosomes for gene mapping studies.

Animals↗
PubMed1988

Expression of hepatitis B surface antigen in human cells by a recombinant BK virus DNA vector.

The construction of the first stable human cell lines that express and secrete authentic hepatitis B virus surface antigen (HBsAg), using a BK virus (BKV) episomal plasmid vector, is described. The amount of HBsAg produced by BKV vectors (up to 600 ng/10(7) cells) was comparable to other eukaryotic vector systems. The level of HBsAg expression remained the same regardless of the orientation of the HBsAg gene, substitution of the HBsAg gene promoter with the mouse metallothionein I gene promoter or the tissue origin of the human cell lines used to establish stable cellular transformants. Northern blot analysis also indicated synthesis of normal HBsAg transcripts. Surprisingly, however, the vectors were maintained at far lower than expected copy number (one to five copies/cell). Reasons for this are discussed.

BK Virus↗