The ends of human chromosomes (telomeres) lose up to 200 bp of DNA per cell division. Chromosomal shortening ultimately leads to senescence and death in normal cells. Many human carcinoma lines are immortal in vitro, suggesting that these cells have a mechanism for maintaining the ends of their chromosomes. Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosomes using its RNA component as template. Telomerase activity is found in most tumor cells, but is absent from normal cells. Little is known about how normal human cells repress telomerase (hTERT) gene expression. Mice carrying an E2F-1 null mutation develop a variety of malignant tumors, suggesting that this transcription factor has a tumor suppressor function. To determine mechanisms by which E2F-1 suppresses tumor formation, we examined the role of this transcription factor in regulation of the hTERT promoter in human cells. We identified two putative E2F-1-binding sites proximal to the transcriptional start site of the hTERT promoter. Mutation of these sites produced dramatic increases in promoter activity. Overexpression of E2F-1 but not a mutant E2F-1 repressed hTERT promoter activity in reporter gene assays. This repression was abolished by mutation of the E2F-1-binding sites in the hTERT promoter. Human cancer cell lines stably overexpressing E2F-1 exhibited decreased hTERT mRNA expression and telomerase activity. We conclude that E2F-1 has an atypical function as a transcriptional repressor of the hTERT gene in human cells.

Tumor cell invasion and metastasis require precise coordination of adherence to extracellular matrix (ECM) and controlled degradation of its components. Invasive cells secrete proteolytic enzymes known as matrix metalloproteinases (MMPs) which degrade specific basement membrane molecules. Expression of these enzymes is regulated by multiple signaling mechanisms, including the mitogen-activated protein kinase (MAPK) pathway. One of the terminal effectors of this signaling cascade is jun N-terminal kinase 1 (JNK1) which phosphorylates the transcription factor c-jun, a component of the AP-1 complex. MMP-9 expression is regulated by two well-characterized AP-1 sites in the promoter of this gene. To determine how JNK1 activity regulated MMP-9 expression in human squamous cell carcinoma lines, we overexpressed this kinase in SCC25 cells. JNK1 overexpression induced MMP-9 protein levels and activity in this cell line. Elevated MMP-9 expression correlated with increased invasion of reconstituted basement membranes by JNK1-overexpressing clones. Site-directed mutagenesis of the MMP-9 promoter revealed that JNK1 cooperated with its transcription factor target c-jun to increase MMP-9 expression at the transcriptional level via the proximal AP-1 site. These results suggest that elevated JNK1 expression may contribute to increased MMP-9 activity and ECM invasion by tumor cells.

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Growth factor receptors of the tyrosine kinase family regulate proliferation of a variety of cell types. In some human cancers, the epidermal growth factor receptor (EGFR) and its ligands often are overexpressed, leading to both constitutive and autocrine activation. Intracellular signaling via this receptor takes place through several mechanisms of action, including activation of ras and the mitogen-activated protein kinase (MAPK) pathway. Our previous studies have shown that human squamous cell carcinoma (SCC) lines overexpress EGFR and do not increase proliferation in response to exogenous epidermal growth factor (EGF). The vitamin A metabolite retinoic acid (RA) has been used as a chemotherapeutic drug in the treatment of SCC. RA decreases proliferation of SCC lines, in part owing to inhibition of EGFR expression. However, we previously found that treatment of SCC lines with inhibitory doses of RA sensitized cells to the proliferative effects of EGF. We now present a mechanism of action for this effect. RA inhibited expression of EGFR and proteins in the MAPK signaling pathway. Expression of these molecules returned to basal levels within 24 h after RA withdrawal. RA also inhibited autocrine secretion of EGF, which returned to basal levels with slower kinetics. During this time, addition of exogenous EGF stimulated mitosis in SCC lines. These data suggested that signaling proteins downstream of overexpressed EGFR may have limited the mitotic response in SCC lines. In support of this hypothesis, overexpression of the EGFR adaptor protein Grb2 increased cell proliferation and restored EGF-induced mitosis.