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W K Roth

Publications and source records attributed to W K Roth.

18 recordsLinked to original sources
PubMed1992

Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance.

As previously described, proliferation of Kaposi sarcoma (KS)-derived cells in vitro is dependent on the presence of platelet-derived growth factor (PDGF). To test the hypothesis that PDGF may also be a major growth factor for KS cells in vivo, we performed in situ hybridization and immunohistochemical staining for PDGF and PDGF receptors in tissue sections of AIDS-related KS. The data suggest that KS consists of two types of tumor cells. (i) The main population are spindle-shaped cells with elongated nuclei (KS-s cells). They reveal a strong expression of PDGF beta receptors but do not express the PDGF-A and PDGF-B isoforms. (ii) A minor population of KS cells express PDGF beta receptor as well as PDGF-A and PDGF-B (KS-p cells). These cells are often grouped in whorls and surrounding vascular slits. They reveal spherical nuclei with evenly distributed chromatin and inconspicuous nucleoli. PDGF alpha receptor is not expressed in either form of KS cells. The results suggest that the isoforms of PDGF and the PDGF beta receptor are differentially expressed in two different cell types in KS and that PDGF isoforms may contribute to the pathogenesis of KS.

Acquired Immunodeficiency Syndrome↗
PubMed1992

'Run-off' polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA hybridization probes: synthesis and application.

In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a 'run-off' reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The 'run-off' reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dot-blot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6.5 x 10(8) cpm micrograms-1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.

Actins↗
PubMed1992

Kaposi's sarcoma: a review of gene expression and ultrastructure of KS spindle cells in vivo.

The ultrastructural features and the gene expression pattern of Kaposi's sarcoma (KS) spindle cells in vivo suggest that KS is a tumor of the mixed cell type. The expression pattern of cytokines and cytokine receptors in the tumor lesion, together with the results obtained from in vitro characterization of KS-derived cells, provide evidence that paracrine mechanisms of growth factor action are important for the maintenance of KS. The reports on virus infection of KS cells suggest an indirect role of virus infection in the induction of KS, most likely mediated by immunostimulation and subsequent production of cytokines.

Biomarkers↗
PubMed1991

HIV-associated Kaposi's sarcoma: new developments in epidemiology and molecular pathology.

New epidemiological data give evidence for an unknown etiological agent of Kaposi's sarcoma (KS). Experimental support is provided by research on cultivated KS cells. These results contradict a direct involvement of HIV-1 in the pathogenesis of KS. Research on cultivated KS cells confirmed the hypothesis that KS spindle cells originate from endothelial cells and gave new insight into the pathogenesis of tumor cell growth. KS spindle cells secrete an autocrine acting growth promoting activity. Nevertheless, they seem to depend on several growth factors like PDGF and IL-6 provided by surrounding endothelial cells and macrophages, respectively. The results support the hypothesis of a tumor relying on paracrine acting factors more than on autocrine acting factors.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Fibroblast growth factor 5 proto-oncogene is expressed in normal human fibroblasts and induced by serum growth factors.

Fibroblast growth factor 5 (FGF-5) is a member of the fibroblast growth factor family with transforming potential. It has been found to be expressed in several human tumor cell lines, but nothing is known about expression of this growth factor in normal cells and its biological functions. Here we show that the FGF-5 gene is expressed in exponentially growing normal human fibroblasts. In quiescent fibroblasts, expression of FGF-5 is strongly induced by serum and several growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). This induction can be mediated by at least two different pathways involving protein kinase C or cAMP-dependent kinases. Since the effect is independent of de novo protein synthesis, FGF-5 represents the product of a primary response gene. In addition our data suggest that FGF-5 is mitogenic for human fibroblasts, indicating the existence of an FGF-5-mediated positive feedback in these cells which could amplify and prolong the cellular response to the initial stimulus.

Animals↗
PubMed1991

[Morphologic HIV demonstration in formalin embedded material--techniques, problems, results].

Formalin-fixed, paraffin-embedded material of archival specimens is suitable for a morphological HIV-detection: Infected cells with HIV in the proliferative phase can be demonstrated with reliable results on tissue sections by immunohistological technics using new antibodies. In situ nucleic acid hybridisation technics can also show HIV in the expression phase on paraffin-embedded material, but often fail in demonstrating latently HIV-infected cells. The DNA-Polymerase chain reaction can detect latent Provirus in morphologically defined areas of paraffin sections even in autopsy material, i.e. lymphnodes and even eyes of patients with HIV-Infection, but requires precaution and control with respect to contamination.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Low mitogenic response to EGF and TGF-alpha: a characteristic feature of cultured Kaposi's sarcoma derived cells.

We have investigated the mitogenicity of Epidermal Growth Factor (EGF) and Transforming Growth Factor alpha (TGF-alpha) for cultured Kaposi's sarcoma derived cells (KS cells). In contrast to control fibroblasts from the same patients, KS cells revealed only a weak mitogenic response to these growth factors. Neither EGF nor TGF-alpha were able to substitute for Platelet-derived growth factor (PDGF) when KS cells were grown in PDGF-depleted Platelet-Poor-Plasma serum (PPPS)-supplemented medium. The low mitogenicity of EGF and TGF-alpha for KS cells is not based upon a reduced expression of EGF receptor mRNA and protein in KS cells. However, the binding of EGF to KS cells is about 50% lower than that to fibroblasts. This reduced binding is not due to an occupation of the receptors by TGF-alpha since the expression level of this mitogen in different KS cell lines does not correlate with their capacity to bind EGF. In contrast, the low EGF binding seems to be an intrinsic feature of the EGF receptors of KS cells. In spite of the low mitogenicity of EGF for the tumor cells, the expression of c-myc, PDGF-A and Fibroblast growth factor 5 (FGF-5) mRNA is equally induced by purified EGF in KS cells and fibroblasts. This shows that at least the signal transduction pathways which lead to the expression of these genes are functional in KS cells.

Blotting, Northern↗
PubMed1990

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.

Animals↗
PubMed1990

Cultured Kaposi's sarcoma-derived cells express functional PDGF A-type and B-type receptors.

We have investigated the role of platelet-derived growth factor (PDGF) for the growth of cultured Kaposi's sarcoma (KS)-derived cells. They were found to bind 125I-PDGF-AA and 125I-PDGF-BB in a manner which is consistent with the presence of both PDGF A-type and PDGF B-type receptors. Furthermore, KS cells responded mitogenically to both PDGF homodimers. This shows that KS cells have functional A- and B-type PDGF receptors, suggesting an important role of PDGF for the growth of KS cells in vitro. In addition we found that KS cells produce PDGF-A chain mRNA in response to PDGF stimulation. This suggests that a PDGF-induced autocrine stimulation involving PDGF-AA which could amplify the response to PDGF occurs in these cells.

Binding, Competitive↗
PubMed1990

Infection of human fibroblasts and osteoblast-like cells with HIV-1.

Primary human skin- and lung-derived fibroblast cell cultures and continuous human osteoblast-like and fibroblast-like cell lines were infected with different strains of HIV-1. Infection was measured at the single-cell level using the immunoperoxidase staining method to detect viral proteins. No cytopathic effects were observed in HIV-1-infected cell cultures. One continuous cell line (LC5), derived from embryonic lung, was readily infectable with HIV-1 and showed continuous production of infectious virus. Infection of LC5 cells could be blocked with anti-CD4 monoclonal antibodies. These findings indicate that fibroblasts of skin and lung, and osteogenic cells may be considered as potential target cells for HIV-1, thereby possibly contributing to the establishment of local HIV reservoirs.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Invasive migration of epidemic Kaposi's sarcoma cells in vitro.

Kaposi's sarcoma (KS) is a low grade malignant neoplasm which shows invasive growth and often occurs in immunosuppressed patients with the Acquired Immune Deficiency Syndrome (AIDS; epidemic KS). It is also found in elderly men where it is usually limited to the skin (classic KS). The present study investigated the chemotaxis and invasive migration of epidemic KS cells in vitro and compared them to cells grown from classic KS lesions and to fibroblasts. Epidemic KS cells demonstrated invasive migration through reconstituted basement membrane (Matrigel) as well as through interstitial connective tissue (collagen I) in early passages, whereas fibroblasts did not invade either barrier. Epidemic KS cells in late passages did not show any invasive migration. Following pretreatment with tumour necrosis factor alpha (TNF-alpha) there was no enhanced migration through the Matrigel and collagen I for epidemic KS cells, whereas classic KS cells showed an increased migration through the type I collagen barrier.

Basement Membrane↗
PubMed1989

Cells derived from sporadic and AIDS-related Kaposi's sarcoma reveal identical cytochemical and molecular properties in vitro.

We have established 40 cell cultures from biopsies of 5 patients with sporadic Kaposi's sarcoma (KS) and compared them with cell cultures derived from KS biopsies of AIDS patients. Immunocytochemical staining of sporadic KS cells revealed the same endothelial cell markers as those expressed on AIDS-KS cells, indicating that both types of KS might be of endothelial cell origin. In contrast to clinical features, in vitro growth properties showed no differences. KS cells of both types reveal a low degree of malignancy but can be distinguished from fibroblasts by a higher passage number in low serum concentrations and a more pronounced dependence on platelet-derived growth factor (PDGF). The DNA of 12 cell lines of both types of KS was negative for genomic equivalents of hepatitis B virus (HBV), cytomegalovirus (CMV) and, in the case of AIDS-KS, for human immunodeficiency virus (HIV). No rearrangements or amplifications of several oncogenes, often involved in human tumors, were detected. The expression of these oncogenes by sporadic and AIDS-KS cells, as analyzed by Northern blotting, was comparable to that of normal dermal fibroblasts from the same patients. Our results indicate that the 2 types of Kaposi's sarcoma possess identical cytochemical and molecular properties. They are probably weakly malignant neoplasms of endothelial cell origin, and paracrine growth stimulation appears to be important for their maintenance and progression.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Cytochemical and molecular properties of simian virus 40 transformed Kaposi's sarcoma-derived cells: evidence for the secretion of a member of the fibroblast growth factor family.

Contact-inhibited Kaposi's sarcoma-derived cells (KS cells) were transfected with Simian Virus 40 (SV40) DNA. Transformed cells (SV-KSC) were selected for their capacity to form foci on monolayers of the low-malignant KS cells. Isolated SV-KSC foci were found to contain integrated SV40 DNA sequences and to express SV40 large T-antigen. Several differentiation properties of KS cells are retained in the SV40 transformants, e.g., expression of vimentin and the endothelial cell marker BMA 120. In contrast to the maternal KS cells, SV-KSC are capable of growing in platelet-derived growth factor (PDGF)-depleted platelet-poor-plasma serum (PPPS) and in soft agar. However, they are not tumorigenic in nude mice. Expression of the oncogenes c-myc, c-N-ras, c-Ha-ras, and p53 is significantly elevated in SV-KSC, whereas c-fos and c-erb B expression is comparable to that of KS cells and fibroblasts. Conditioned medium from SV-KSC can substitute for PDGF when PDGF-dependent, nontransformed KS cells are grown in PPPS. Biochemical analysis of the SV-KSC supernatant and PDGF A and B mRNA expression analysis provide evidence that the mitogenic activity is not due to a PDGF-like growth factor. On the other hand, there is evidence to indicate that the SV-KSC mitogen is a member of the fibroblast growth factor family. SV-KSC represent an interesting model system for the study of different degrees of malignancy of cultured mesenchymal cells and especially provide an important source for the isolation of a potent growth factor for KS cells and other mesenchymal cells in vitro.

Cell Line, Transformed↗
PubMed1989

Depletion of PDGF from serum inhibits growth of AIDS-related and sporadic Kaposi's sarcoma cells in culture.

We have examined the mitogenic potential of platelet-derived growth factor (PDGF) on AIDS (acquired immune deficiency syndrome)-related and sporadic Kaposi's sarcoma cells in comparison to fibroblasts at physiological and subphysiological calcium concentrations of the culture medium. At low calcium concentrations in the presence of 3% human serum the growth rate of fibroblasts and Kaposi's sarcoma (KS) cells is similarly reduced to less than half of the growth rate at physiological Ca2+ concentrations. In the presence of 3% PDGF depleted, platelet poor plasma-derived serum (PPPS) growth of KS cells ceased completely, whereas fibroblasts made 1-2 cell divisions within 15 days. At physiological Ca2+ concentrations, the reduced PDGF content in 3% PPPS had no effect on human embryonal fibroblasts and little effect on adult skin fibroblasts. In contrast, KS cells became growth-arrested after one to two doublings. This is consistent with the observation that PDGF B-chain mRNA could not be detected in our KS cells whereas PDGF receptor mRNA was expressed.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Cultured, AIDS-related Kaposi's sarcoma cells express endothelial cell markers and are weakly malignant in vitro.

Up to 30% of patients with acquired immunodeficiency syndrome (AIDS) suffer from Kaposi's sarcoma (AIDS-KS). The histogenesis and neoplastic nature of this tumor is still controversial. We have established cell cultures of KS biopsies from 7 patients with AIDS. All donors were seropositive for the human immunodeficiency virus I (HIV-I), cytomegalovirus (CMV) and hepatitis B virus (HBV). The tumors were histologically shown to be KS. Cell cultures derived from these tumors all expressed the endothelial cell marker BMA 120 antigen. Most of these cultures were positive for acetylated low-density lipoprotein (acLDL) uptake and alkaline phosphatase (AP) expression, and negative for factor-VIII-related antigen (FVIII-RAg). The staining pattern was heterogeneous with respect to number of endothelial cell markers expressed in each culture. We conclude from subcloning experiments that the cultured cells cease to express acLDL receptor and AP, but not the antigen detected by the monoclonal antibody (MAb) BMA 120. The cells grew well in culture up to 50 passages and showed a fibroblast-like morphology. Assays performed to investigate their degree of malignancy revealed a significantly increased passage number under reduced serum conditions as compared to normal fibroblasts but no tumor formation in nude mice. Neither HIV, HBV nor CMV sequences were found in any of the cell lines tested. We conclude that AIDS-KS is an endothelial-cell-derived neoplasm of low malignancy and that HIV, HBV and CMV are not directly involved in its genesis.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.

A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.

Chelating Agents↗