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J S Rhim

Publications and source records attributed to J S Rhim.

At least 19 recordsLinked to original sources
PubMed1992

Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.

We have used antisense RNA technology to inhibit endogenous parathyroid hormone-related peptide (PTHRP) production in the established human keratinocyte cell line, HPK1A, in order to assess the role of PTHRP as a potential modulator of cell growth. Rat PTHRP cDNA was cloned into the replication defective retroviral vector pYN in an antisense orientation and a stable cell line (HPK1A-AS) was generated after infection by amphotropic virus and selection by the neomycin derivative, G418. Expression of the transfected antisense sequence was confirmed with an RNA sense probe for PTHRP. The effect of the retrovirally mediated gene transfer on the endogenous PTHRP transcript was examined with an RNA antisense probe which demonstrated an absence of the endogenous transcript in HPK1A-AS cells. A 1.6-kilobase transcript was, however, present in equivalent quantities in both uninfected HPK1A and pYN-infected (HPK1A-pYN) cells. Immunocytochemistry and assessment of PTHRP secretion into the medium using an NH2-terminal radioimmunoassay and a UMR 106 adenylate cyclase bioassay confirmed the absence of PTHRP in HPK1A-AS cells. Examination of the inhibition of PTHRP production on cell growth demonstrated a reduction in doubling time and an increase in [3H]thymidine incorporation. Cell cycle analysis showed an increase in the proportion of the cell population in the S phase (relative to G0/G1) in HPK1A-AS cells compared to HPK1A or HPK1A-pYN cells. These data, therefore, indicate that endogenous PTHRP acts as an effective inhibitor of cell growth in this keratinocyte model and that this action occurs, at least in part, by diminishing entry into the S phase of the cell cycle. Furthermore, the antisense RNA method is a potent one to evaluate the cellular actions of PTHRP.

Amino Acid Sequence↗
PubMed1992

Neoplastic transformation of immortalized human keratinocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most powerful carcinogen ever tested in animals. Recent epidemiological studies have suggested its carcinogenic potential in humans. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12-simian virus 40 (Ad12-SV40) were transformed by exposures of TCDD equal to or greater than 0.1 nM for 2 wk. These transformed cells showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas no such transformation phenotypes were observed with exposures of less than 0.1 nM for 2 wk. Primary human epithelial keratinocytes exposed to various concentrations of TCDD failed to show any evidence of transformation. Induction of aryl hydrocarbon hydroxylase activity was dose dependent, as was transformation. Thus, the carcinogenicity of TCDD in this human cell system appears to be an Ah receptor-mediated process. The present study represents the first evidence of neoplastic conversion of human cells exposed to this environmentally important chemical.

Animals↗
PubMed1992

Role of DNA repair in malignant neoplastic transformation of human mammary epithelial cells in culture.

Epithelial cells derived from normal human mammary tissue were examined for capacity to repair radiation-induced chromatin DNA damage. Repair capacity was estimated by quantifying chromatid aberrations in metaphase cells arrested 0.5-1.5 h after X-irradiation during G2. The parental cells at passage 12 had 19 chromatid breaks and 16 gaps per 100 metaphase cells, representing efficient repair. Of two continuous cell lines, derived after benzo[a]pyrene treatment, A1 maintained the efficient repair phenotype through passage 50, while a subline of A1 developed the repair-deficient phenotype characterized by a 3- to 5-fold higher frequency of chromatid breaks or gaps. This line was transformed to tumorigenic cells by HaMSV and SV40 T antigen. The second continuous line B5 and derivatives had 102-165 chromatid breaks and 87-134 gaps per 100 metaphases (deficient repair phenotype). This line was transformed to tumorigenic cells by KiMSV. As reported previously for human epidermal keratinocytes, acquisition of this repair-deficient phenotype appears to be an early requisite step in the malignant neoplastic transformation of human cells in culture.

Adult↗
PubMed1992

Identification and functional characterization of adenylate cyclase-linked receptors for parathyroid hormone-like peptides on immortalized human keratinocytes.

We have identified and characterized receptors for the amino-terminal domains of PTH and PTH-like peptide (PLP) on an immortalized human keratinocyte cell line, RHEK-1. Binding of both PLP-(1-34) and PTH-(1-34) to the RHEK-1 cells was consistent with a two-site model; affinities and capacities for each site were similar for the two peptides. Both peptides also stimulated adenylate cyclase activity with an equal ED50 in this cell line. Pertussis toxin pretreatment enhanced this peptide-mediated enzyme activity, suggesting linkage of the receptor to an inhibitory guanyl nucleotide-binding protein (Gi). Adenylate cyclase activity was diminished by both homologous [PLP-(1-34)] and heterologous [epidermal growth factor (EGF)] effectors. Malignant conversion of the immortalized cells with an activated H-ras oncogene to produce the RHEK-ras cell line was associated with a reduction in binding at both PLP/PTH and EGF receptors as well as a postreceptor defect in PLP/PTH-stimulated adenylate cyclase activity. The defect in enzyme activity appeared to be due in part to a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs), but not to an increase in Gi activity. Activation of the keratinocyte amino-terminal PLP/PTH receptor resulted in a small increase in [3H]thymidine incorporation, which was associated with an increase in cell numbers. This mitogenic effect was enhanced in the presence of EGF and was markedly reduced when cells were cultured in a high extracellular calcium environment. These studies demonstrate that the amino-terminal region of PLP and PTH activates adenylate cyclase-linked receptors, which are associated with mitogenesis, in RHEK-1 cells and suggest that this cell line represents a suitable model in which to examine the actions of PLP in keratinocytes.

Adenylyl Cyclases↗
PubMed1992

The HIV tat gene transforms human keratinocytes.

Skin disorders are frequently seen in patients with the acquired immune deficiency syndrome (AIDS). Since many of these cutaneous manifestations are accompanied by an early onset of epidermal hyperplasia, the keratinocyte is a candidate for infection by the human immunodeficiency virus (HIV). We now report that the HIV tat gene, under the control of the viral long terminal repeat (LTR), can efficiently transform human keratinocytes in culture. Our finding suggests that this activity of the tat gene may be responsible for the epidermal hyperplasia that accompanies psoriasis and precedes the development of squamous cell and basal cell carcinomas in AIDS patients.

Animals↗
PubMed1991

Malignant human papillomavirus type 16-transformed human keratinocytes exhibit altered expression of extracellular matrix glycoproteins.

We found that keratinocytes immortalized with human papillomavirus (HPV) type 16 DNA and malignantly converted by H-ras transfection (HPK-1A/ras) exhibit an enhanced ability to synthesize a fibronectin-containing extracellular matrix. Gene expression of fibronectin and thrombospondin was increased in tumorigenic keratinocytes compared to control and immortalized keratinocytes, 6- and 9-fold, respectively. Increased production of soluble and cell surface-associated fibronectin was not specific for HPV 16 transformed keratinocytes. Ad12-SV40-immortalized keratinocytes malignantly converted by H-ras transfection (RHEK-1/ras) also exhibited enhanced expression of fibronectin and thrombospondin, as well as pro-alpha 1 type I collagen. Steady state mRNA levels for autocrine growth-regulatory factors, transforming growth factors alpha and beta 1, were increased in Ad12-SV40 but not HPV 16-transformed human keratinocytes. We then determined whether increased production of fibronectin was associated with aberrant differentiation of transformed keratinocytes. Less than 10% of the HPV 16-transformed cells produced cornified envelopes after suspension-induced differentiation compared to 70% of normal keratinocytes. However, immortalization by HPV 16 DNA was sufficient to confer a differentiation-defective phenotype. Both involucrin mRNA and protein levels were decreased 8-fold in HPV 16-immortalized keratinocytes compared to normal cells and malignant conversion further attenuated involucrin levels. These studies demonstrate that aberrant differentiation is an early event in the transformation of the human keratinocytes and is not the result of enhanced expression of the extracellular matrix proteins. Unlike transformed fibroblastic cell types, up-regulation of fibronectin gene expression and matrix formation is a consistent characteristic of malignantly converted human keratinocytes.

Cell Differentiation↗
PubMed1991

Current state-of-the-art in human cell transformation in culture.

The immortalization and transformation of cultured human cells has far-reaching implications for both cell and cancer biology. Human cell transformation studies will increase our understanding of the mechanisms underlying carcinogenesis and differentiation. The neoplastic process can now be studied in a model human cell culture system. The accompanying biochemical and genetic changes, once identified, will help define the relationship between malignancy and differentiation. The present studies indeed demonstrate that the neoplastic process can now be studied in a human cell model system. Primary human cells treated with various carcinogens became immortalized in culture but were not tumorigenic. Additional exposure to either retroviruses, chemical carcinogenes or X-ŗay irradiation to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary cells in culture by the combined action of either a DNA transforming virus and a retrovirus or a DNA virus and a chemical or X-ray irradiation, and support an multistep process for neoplastic conversion. It has been known that normal human cells in culture are remarkably resistant to experimentally induced tumorigenicity. However, as shown above, normal human cells could now be transformed into tumorigenic cells.

Cell Transformation, Neoplastic↗
PubMed1990

Evidence for the multistep nature of in vitro human epithelial cell carcinogenesis.

In keeping with the multistep development of human cancer in vivo, a stepwise approach to neoplastic transformation in vitro presents a reasonable strategy. We have recently developed an in vitro multistep model suitable for the study of human epithelial cell carcinogenesis. Upon infection with the adenovirus 12-simian virus 40 hybrid virus, primary human epidermal keratinocytes acquired an indefinite life span in culture but did not undergo malignant conversion. Subsequent addition of Kirsten murine sarcoma virus and human ras oncogene or chemical carcinogens (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline 1-oxide) to these cells induced morphological alterations and the acquisition of neoplastic properties. Subsequently it was found that this line could be transformed neoplastically by a variety of retrovirus-containing H-ras, bas, fes, fms, erbB, and src oncogenes. In addition, we found that the immortalized human epidermal keratinocyte (RHEK-1) line can be transformed neoplastically by exposure to ionizing radiation. Thus, this in vitro system may be useful in studying the interaction of a variety of carcinogenic agents and human epithelial cells. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of viruses, oncogenes, chemical carcinogens, or X-ray irradiation and support a multistep process for neoplastic conversion.

Adenoviridae↗
PubMed1990

Morphological transformation of human keratinocytes expressing the LMP gene of Epstein-Barr virus.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been known for some time, but the precise role of EBV in this cancer is poorly understood, due partly to the lack of an in vitro system for studying NPC cells and the effect of EBV on epithelial cells. Biopsies of NPC tumours have revealed expression of the EBV latent membrane protein (LMP) in 65% of cases, suggesting that in at least some NPC tumours LMP may contribute to cell transformation. Here we address the question of the effect of LMP expression on epithelial cells. Transfection of an immortalized, non-tumorigenic keratinocyte cell line (RHEK-1) with the LMP gene causes a striking morphological transformation: the originally flat, polygonal colonies change to bundles of spindle-shaped cells that form multilayer foci, and cytokeratin expression is down-regulated. Our results suggest that LMP expression may be an important causal factor in the development of NPC.

Antigens, Viral↗
PubMed1990

TPR-MET oncogenic rearrangement: detection by polymerase chain reaction amplification of the transcript and expression in human tumor cell lines.

Activation of the MET protooncogene by a rearrangement involving the fusion of TPR and MET specific gene sequences has been observed in a human osteosarcoma cell line (HOS) treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No information has been available about the possible occurrence of this rearrangement in human tumors. To facilitate rapid screening of human cell lines and tumor samples for this specific gene rearrangement, we developed a sensitive detection method based on polymerase chain reaction (PCR) amplification of TPR-MET mRNA. cDNA was generated from cellular transcripts by using one of the PCR primers, which was then used as a template for PCR amplification of a 205-base-pair region carrying the breakpoint. An end-labeled internal probe was hybridized in solution to an aliquot of the PCR product for detecting amplification. Cells could be directly screened by the assay without prior isolation of RNA. A 205-base-pair DNA fragment characteristic of the TPR-MET rearrangement was detected in cell lines previously known to contain this altered sequence. The rearrangement was also detected at very low levels in the parental (nontransformed) cell line, HOS TE-85. A preliminary survey of cell lines derived from a variety of human tumors indicates that TPR-MET rearrangement occurred and was expressed at very low frequencies by cells from 7 of 14 tumors of nonhematopoietic origin.

Animals↗
PubMed1990

Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation.

Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.

Animals↗
PubMed1990

CR2 complement receptor.

CR2, a membrane glycoprotein, is one of a number of cell-surface proteins which bind activation and processing fragments of the complement system. CR2, which is found on normal B lymphocytes, follicular dendritic cells in lymphoid organs, and epithelial cells, interacts preferentially with C3dg, the terminal activation/processing fragment of the third complement component. Attachment of C3dg to CR2 brings complement activators, bearing covalently bound C3dg, into direct membrane contact with CR2-bearing cells. Epstein-Barr virus, a human herpesvirus, also binds to CR2 on B lymphocytes. Attachment of EBV is followed by infection. CR2 has been purified and the binding properties of its ligands analyzed. Monoclonal antibodies have been developed and used to probe the structural correlates of CR2 functions. CR2 has been molecularly cloned and its primary amino acid sequence deduced. These data indicate that it shares characteristic structural features with a number of other complement and non-complement cell membrane and plasma proteins. Several of the complement-associated proteins in this family possess regulatory functions; they are encoded by linked genes which have been localized to band q32 on chromosome 1. CR2 has been expressed in primate and rodent cells by transfection of cDNA in antigenically and functionally intact form. It has also been expressed in soluble form and its structure, electron microscopic appearance and binding characteristics analyzed in detail. The present state of knowledge of the structure and genetics of CR2 and current understanding of its biologic functions are summarized here.

Animals↗
PubMed1989

Amplification, overexpression, and rearrangement of the erbB-2 protooncogene in primary human stomach carcinomas.

Four of 51 primary gastric carcinomas exhibited amplification of the erbB-2 protooncogene ranging from 2- to 8-fold. In three cases gene amplification affected erbB-2 alleles of normal gene structure as determined by Southern blot analysis. In addition, one tumor displayed gene amplification of an apparently rearranged erbB-2 allele. Analysis of the rearranged allele revealed a structural alteration consistent with an internal deletion of approximately 2 kilobase pairs within the erbB-2 gene. Quantitation of erbB-2 mRNA in these tumors demonstrated that gene amplification coincided with overexpression of erbB-2 mRNA ranging from 8- to 32-fold above levels observed in stomach tumors without gene amplification. Furthermore, in one of the tumors with amplified normal size erbB-2 restriction fragments, elevated erbB-2 mRNA levels consisted of the normal size 5-kilobase transcript. Thus, overexpression of normal size erbB-2 mRNA accompanies gene amplification in primary stomach tumors. In addition, evidence for erbB-2 gene rearrangement suggests a role for such alteration in the development of certain gastric carcinomas.

Blotting, Northern↗
PubMed1989

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients.

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.

Cell Line↗
PubMed1989

Glucocorticoid-enhanced neoplastic transformation of human keratinocytes by human papillomavirus type 16 and an activated ras oncogene.

Human papillomaviruses (HPV) are known etiological agents of benign proliferation of the skin and mucosa (papillomas and warts). They have also been implicated in the development of cervical dysplasia and anogenital carcinoma. The close association of HPV type 16 DNA with the majority of cervical carcinomas suggests the involvement of the virus in this type of cancer. We have developed an in vitro multistep model for human epithelial cell carcinogenesis. Primary human epidermal keratinocytes acquired an indefinite lifespan in response to transfection with HPV 16 DNA but did not undergo malignant conversion. Addition of Kirsten murine sarcoma virus (Ki-MSV), which contains an activated K-ras oncogene, to these cells induced morphological alterations associated with the acquisition of neoplastic properties. The frequency of transformation by Ki-MSV was markedly enhanced by the inclusion of glucocorticoid. At optimal conditions, a 125-fold stimulation was observed. These findings demonstrate the malignant conversion of human primary epithelial cells in culture by the cooperation of HPV type 16 and an activated ras oncogene and support a multistep process for neoplastic conversion. The availability of a human epithelial cell transformation model should facilitate studies of the interaction between HPV and human epithelial cells.

Blotting, Western↗
PubMed1989

Adenosine-sensitive phosphoinositide turnover in a newly established renal cell line.

To aid in characterizing adenosine receptors in renal cells, primary cultures of rabbit cortical collecting tubule (RCCT) cells were infected with an adenovirus 12-simian virus 40 hybrid, resulting in a continuous cell line. The cells, designated RCCT-28A, retained their epithelial morphology and reacted with a monoclonal antibody specific for rabbit collecting tubule. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was stimulated by vasopressin (AVP), isoproterenol, prostaglandin E2 (PGE2), calcitonin, parathyroid hormone, and a potent adenosine A1- and A2-receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA). A more selective adenosine A1-receptor agonist, N6-cyclohexyl adenosine (CHA) inhibited basal and AVP-stimulated cAMP accumulation. Cytosolic free calcium was transiently elevated by bradykinin, PGE2, NECA, and CHA. To examine the mechanism by which adenosine analogues increase intracellular free calcium, phosphoinositide (PI) turnover was assessed in the 28A cells after labeling with myo-[3H]inositol. NECA and CHA increased [3H]inositol phosphate formation with an approximate half-maximal effective concentration of 0.1 microM for both analogues. The increase in PI turnover was blocked by the selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine and pretreatment of the 28A cells with pertussis toxin. These results suggest that adenosine analogues increase cytosolic free calcium by stimulating PI turnover.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Neoplastic transformation of human keratinocytes by polybrene-induced DNA-mediated transfer of an activated oncogene.

Polybrene, in conjunction with dimethyl sulfoxide (DMSO) shock has been shown to increase the frequency of DNA-mediated gene transfer to mammalian cells as compared with the frequency obtained with calcium phosphate transfection. We have successfully adapted this procedure for use with human epidermal keratinocytes. Non-tumorigenic human epidermal epithelial cells immortalized by SV40 tumor antigen were neoplastically transfected, using Polybrene at a concentration of 10 micrograms ml-1, followed by a 4 min shock, with 30% DMSO, with a plasmid carrying the activated H-ras gene from the EJ bladder carcinoma cell line. The transfected cells showed morphological alterations and induced carcinomas when transplanted into nude mice. They contained integrated copies of the transfected H-ras gene and expressed high levels of the p21 protein. Polybrene-induced DNA transfection, therefore, offers the opportunity to transfer genes effectively into human epidermal keratinocytes and should accelerate the study of the interaction between oncogenes and human epithelial cells. This study appears to represent the first neoplastic conversion of nontumorigenic, immortalized human epidermal keratinocytes by an activated human oncogene.

Animals↗