Yao Qi Huang

Human herpesvirus-like nucleic acid in various forms of Kaposi's sarcoma

The association between a new human herpesvirus-like agent and various forms of Kaposis sarcoma was examined by PCR. The DNA sequences of this agent were detected in 7 of 8 classic Kaposis sarcoma specimens, 12 of 12 AIDS-associated specimens from the United States, and 7 of 10 specimens from African endemic Kaposis sarcoma. Polymorphism of the herpesvirus-like DNA in the Kaposis tissue from different populations was observed by both single-strand conformational polymorphism and direct sequencing. Furthermore, the presence and expression of the virus was detected in some Kaposis tumours by Southern and northern blotting. This herpesvirus may be involved in the pathogenesis of different kinds of Kaposis sarcoma seen among distinct and unrelated populations.

HPV-16-related DNA sequences in Kaposi's sarcoma.

In the USA, Kaposis sarcoma associated with the acquired immunodeficiency syndrome (AIDS-KS) is ten times more common in homosexual or bisexual men than in heterosexual men with AIDS. One explanation for this finding is that AIDS-KS may be caused by an infectious agent. Because there is a high incidence of human papillomavirus (HPV) infection, especially HPV-16, in homosexual men, we have sought HPV DNA sequences in Kaposis sarcoma. We used the polymerase chain reaction with a primer pair specific for the highly conserved E6 region of HPV-16 to detect HPV-16 homologous DNA fragments in tumour tissues from 97 patients with KS and in KS-derived cell cultures. HPV DNA sequences were found in 11 of 69 KS skin tumours from homosexual men with AIDS-KS, in 3 of 11 KS biopsy specimens from homosexual men who had no clinical or laboratory evidence of HIV-infection, and in 5 of 17 KS skin lesions from HIV-1-negative elderly men and women with classic KS. The same primer pair amplified HPV-16 homologous fragments from two different continuous cell cultures derived from pleural effusion fluid of patients with pulmonary AIDS-KS and two continuous cell cultures derived from KS skin lesions. The findings suggest that HPV-16-related DNA sequences are associated with different forms of KS and may have a role in the pathogenesis of this neoplasm.

Transcription of human herpesvirus-like agent (HHV-8) in Kaposi's sarcoma.

Recently, DNA sequences of what appear to be a unique human herpesvirus-like agent (HHV-8) have been detected in different types of Kaposis sarcoma (KS) tumors (Chang, Y., E.C. Cesarman, M.S. Pessin, F. Lee, J.C. Culpepper, D.M. Knowles, and P.S. Moore. 1994. Science (Wash. DC). 266:1865-1869). To further elucidate the possibility that HHV-8 plays a role in the pathogenesis of KS, the expression of HHV-8 RNA was examined in fresh KS tissue specimens which were found to harbor HHV-8 DNA by polymerase chain reaction (PCR). The transcription of HHV-8 RNA was detected by RT-PCR in 26 of 29 specimens (89.7%) of the KS tumors including 2 of 3 CKS and 24 of 26 AIDS-KS. No positive signal was detected in eight biopsy specimens of normal skin from healthy donors. By Northern blot analysis, the expression of HHV-8 was detected in 2 of 10 KS tumors examined. Furthermore, the RNA transcripts were observed in endothelial cells lining the irregular vascular spaces and perivascular spindle-shaped cells histologically characteristic of KS in 2 out of 8 different KS specimens examined by in situ hybridization using an antisense probe specific of HHV-8. The detection of RNA expression of HHV-8 in KS tumors further supports the possible etiopathogenic role of this virus in the development of KS.

Expression of int-2 oncogene in Kaposi's sarcoma lesions.

Fibroblast growth factors (FGFs), such as basic FGF, have been implicated in the growth of Kaposis sarcoma (KS) cells in vitro. In the evaluation of the expression of the various genes of the different members of the FGF family and their receptors in fresh KS tissue specimens, int-2 was found to be expressed in more than half of the KS tumors examined. Using reverse transcription PCR, the expression of int-2 was detected in 21 of 38 (55.2%) fresh KS biopsy specimens. In contrast, int-2 mRNA transcripts were not found in normal appearing skin from the same patients except in one sample which was obtained from an AIDS patient with disseminated KS lesions. Sequence data confirmed that the amplified sequences were derived from int-2 mRNA with proper splicing. In addition, 12 nucleic acid alterations were identified in eight out of nine KS tumor samples sequenced. Using immunohistochemical methods, int-2 protein was detected in some of the spindle-shaped tumor cells surrounding the abnormal endothelial-lined vascular slits histologically characteristic of KS. Int-2 specific immunostaining was shown to be present in both the nuclei and cytoplasm of these spindle cells but was more pronounced in the nuclei. Neither amplification nor gross rearrangement of the int-2 gene was detected in KS lesions by Southern blot analysis. These results suggest that the expression of int-2 may play a role in the pathogenesis KS by stimulating local angiogenesis and cell proliferation.

HIV-1 infection and modulation of cytokine and growth factor expression in Kaposi's sarcoma-derived cells in vitro.

ObjectivesHIV-1 transcripts have been detected in AIDS-related Kaposis sarcoma (KS) tissues within the factor Xllla + dermal dendrocytes present in the tumor. Various cytokines and growth factors have been shown to influence the growth of KS-derived cells in vitro. HIV-1 preferentially infects CD4 + cells and has also been found to infect some CD4− cells in vitro. The susceptibility of cultured KS cells in vitro to infection with HIV-1 and the expression of interleukin (IL)-1β, IL-6 and basic fibroblast growth factor (bFGF) after exposure to HIV-1 was examined. MethodsThe susceptibility of two different KS-derived cell cultures to HIV-1 infection was examined by the expression of p24 antigen, detection of proviral sequence and electron microscopy. The expression of IL-1β, IL-6 and bFGF was detected by enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. ResultsKS-derived cells can be infected by HIV-1 in vitro. Both KS-derived cells were found to express CD4 mRNA. The expression of IL-1β and IL-6 was increased, whereas the expression of bFGF was not stimulated after exposure of KS cells to HIV-1. ConclusionThese experiments describe the in vitro infection of KS-derived cells by HIV-1 and the expression of various cytokines and growth factor following infection. The increased production of cytokines observed following such infection may be involved in the pathogenesis of AIDS-related KS.

Evaluation of the tumorigenic and angiogenic potential of human fibroblast growth factor FGF3 in nude mice

Abstract Recently, the expression of fibroblast growth factor 3 (FGF3) was found in 55% of human Kaposis sarcoma (KS) tumor tissues examined, while almost no expression of FGF3 was found in normal skin. To further these studies, human FGF3 cDNA were constructed by the overlap-extension method. The proteins translated from two FGF3 cDNA, which differ only in the sequences preceding the AUG presumed to be the initiation codon, were shown to have the same molecular mass. This result suggests that translation of human FGF3, which is different from mouse FGF3, begins only at the AUG site. The human FGF cDNA was transfected into NIH3T3 cells. The NIH 3T3 cells transformed by FGF3 were then injected subcutaneously into athymic nude mice. Nodular lesions developed at the injection sites in all seven mice injected with the F3-1 cell clone, which showed high expression of FGF3, and in two out of six mice injected with the F3-2 cell clone, which expressed a low level of FGF3. Histopathological features of these tumors contained fascicles of spindle-shaped cells surrounding irregular endothelial lined vascular clefts, similar to those observed in human KS lesions. Immunohistochemical staining for factor V111 antigen revealed reactivity in multiple areas, especially in abundant vascular structures of the tumor sections examined. The expression of FGF3 together with the FGF receptors FGFR1, FGFR2, and FGFR3, was detected in the mouse tumors by Northern blot analysis. Our results indicate that tumors induced by FGF3-transformed NIH3T3 cells show some similarities to human KS tumors. In conclusion, our results demonstrate the potential tumorigenic and angiogenic role of human FGF3.

The absence of Tat sequences in tissues of HIV-negative patients with epidemic Kaposi's sarcoma.

ObjectiveTat, an essential regulatory protein of HIV, acts as a growth factor for Kaposis sarcoma (KS)-derived cells in culture. We tested the hypothesis that HIV-negative epidemic KS patients who are also at high risk for HIV disease might have been infected with a defective HIV-1 virus that retained the ability to express Tat. MethodsWe evaluated the presence of Tat sequences in KS tissue and peripheral blood mononuclear cells (PBMC) of HIV-1-negative individuals with epidemic KS who had risk factors for HIV infection by polymerase chain reaction using specific primers for the Tat region of HIV-1. ResultsNo evidence for the presence of Tat-1 sequences or for Tat-expressing defective HIV-1 virus was found. ConclusionThese results suggest that HIV-1 Tat does not play a role in the initiation of KS in HIV-1-negative individuals. Tat might play an indirect role in epidemic KS in HIV-infected patients.