Malignant human papillomavirus type 18 (HPV18)-positive cervical carcinoma cells can be reverted to a nonmalignant phenotype by generation of somatic cell hybrids with normal human fibroblasts. Although nontumorigenic hybrids, their tumorigenic segregants, and the parental HeLa cells have similar in vitro properties, inoculation only of nontumorigenic cells into nude mice results in a selective suppression of HPV18 transcription which precedes cessation of cellular growth. Our present study, aimed at understanding the differential regulation in vitro and in vivo, shows that the JE gene, encoding the monocyte chemoattractant protein (MCP-1), is expressed only in nontumorigenic hybrids. Although the gene, including its regulatory region, is intact, no JE (MCP-1) mRNA is detected in the tumorigenic segregants and in other malignant HPV-positive cervical carcinoma cell lines. Tests of several monocyte-derived cytokines showed that only tumor necrosis factor alpha strongly induces the JE (MCP-1) gene in nontumorigenic cells and that this is accompanied by a dose-dependent reduction of HPV transcription. The JE (MCP-1) up-regulation occurs within 2 h and does not require de novo protein synthesis. The response to tumor necrosis factor alpha seems to be mediated by an NF-KB-related mechanism, since the induction can be completely abrogated by pretreating the cells with an antioxidant such as pyrrolidine dithiocarbamate. Interestingly, cocultivation of nonmalignant hybrids with monocyte-enriched fractions from human peripheral blood also results in an induction of the JE (MCP-1) gene and a concomitant suppression of HPV18 transcription. Neither effect is observed in malignant cells. These data suggest that JE (MCP-1) may play a pivotal role in the intercellular communication by triggering an intracellular pathway which negatively interferes with viral transcription in HPV-positive nontumorigenic cells.
Considering the involvement of a redox-regulatory pathway in the expression of human papillomaviruses (HPVs), HPV type 16 (HPV-16)-immortalized human keratinocytes were treated with the antioxidant pyrrolidine-dithiocarbamate (PDTC). PDTC induces elevated binding of the transcription factor AP-1 to its cognate recognition site within the viral regulatory region. Despite of increased AP-1 binding, normally indispensable for efficient HPV-16 transcription, viral gene expression was selectively suppressed at the level of initiation of transcription. Electrophoretic mobility supershift assays showed that the composition of the AP-1 complex, predominantly consisting of Jun homodimers in untreated cells, was altered. Irrespective of enhanced c-fos expression, c-jun was phosphorylated and became primarily heterodimerized with fra-1, which was also induced after PDTC incubation. Additionally, there was also an increased complex formation between c-jun and junB. Because both fra-1 and junB overexpression negatively interferes with c-jun/c-fos trans-activation of AP-1responsive genes, our results suggest that the observed block in viral transcription is mainly the consequence of an antioxidant-induced reconstitution of the AP-1 transcription complex. Since expression of the c-jun/c-fos gene family is tightly regulated during cellular differentiation, defined reorganization of a central viral transcription factor may represent a novel mechanism controlling the transcription of pathogenic HPVs during keratinocyte differentiation and in the progression to cervical cancer.
The glycolytic pathway inhibitor 2‐deoxyglucose (2‐DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18 (HPV 18) in cervical carcinoma cells and derived non‐tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down‐regulation is selective, since other reference genes are not affected or even up‐regulated under the same experimental conditions. Moreover, 2‐DG appears to restore the normal half‐life of the tumor suppressor gene product p53, because the protein is strongly up‐regulated after HPV18 E6/E7 suppression. The observed 2‐DG‐effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV18 suppression by 2‐DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB‐8, indicating that Ca2+, a known intracellular “second messenger”, is involved in this process. Elevated c‐myc and p53 expression appears to be responsible for the time‐dependent accumulation of apoptotic cells after prolonged 2‐DG treatment. The finding that 2‐DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a pecularity of growing tumors, but even may be an essential prerequisite for the maintenance of virus‐specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2‐DG or other glucose derivatives in the treatment of precancerous and malignant HPV‐associated lesions. Int. J. Cancer 76:000–000, 1998.© 1998 Wiley‐Liss, Inc.
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