Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined, by single-strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen-induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single-base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C----C:G or C:G----G:C transversions. Mutations at codon 61 of the c-Ha-ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c-Ha-ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) induces gene transcription, a process that requires binding of the activated aryl hydrocarbon receptor (AhR) to dioxinresponsive elements (DREs) within the enhancer region of responsive genes. Most of what is known about the molecular mechanism of AhR-dependent gene activation results from studies on the murine prototype TCDD-responsive gene cytochrome P4501A1 (CYP1A1). Much less is known, however, about the regulation of human TCDD-responsive genes. We have therefore conducted a detailed analysis of the enhancer region of the human CYP1A1 gene. From the ten DRE core motifs investigated within a stretch of 1400 bp in two human tumor cell lines using a ligation-mediated PCR technique, five motifs displayed a TCDD-inducible in vivo footprint. Four of these sites were functional enhancer sequences as demonstrated by a transient expression assay. Based on these data, a distinct functional consensus sequence for DRE motifs within the human CYP1A1 gene is suggested. After introduction of the four functional sites into various mouse hepatoma cell lines, only three exhibited a functional response, suggesting some species differences in CYP1A1 gene regulation. In addition to the footprints at DRE sites, we also detected protein-DNA interactions at three G-rich domains located within the enhancer region of the human CYP1A1 gene. Our data show that, besides some similarities in the regulation of the human and mouse CYP1A1 genes, there also exist some distinct differences, including number, location, and functional consensus sequences of DRE motifs, as well as quantity and location of footprinted G-rich domains.Keywords : CYP1A1; 2,3,7,8-tetrachlorodibenzo-p-dioxin ; Ah receptor; dioxin-responsive elements; gene regulation.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a prototype for a large class of halogenated aromatic hydrocarbons that are widespread and persistent environmental contaminants. In rodents, dioxin produces a broad spectrum of toxic responses and is a potent tumor-promoting agent, while its relevance as a toxicant in humans is less clear (for review see [1]). The diversity of effects are assumed to reflect the ability of TCDD to alter gene expression in a species-specific and tissue-specific manner [2Ϫ4]. Many of the actions of TCDD have been shown to be mediated through an intracellular binding protein, designated the Ah receptor (AhR). This receptor is a member of a distinct class of basic helix-loop-helix (bHLH) transcription factors [5]. The inactive Ah receptor resides in the cytoplasm of target cells in a complex with heat-shock protein Hsp90 and possibly other proteins [6Ϫ9]. Upon binding of a ligand, the receptor is released from Hsp90, and heterodimerizes with a second bHLH protein named Ah-receptor nuclear translocator or ARNT [10]. The resultant AhR/ARNT/ligand complex is able to bind to specific DNA enhancer sequences, termed dioxin-responsive elements (DREs), located in the 5′ regulatory region of respon...

Tumor tissue oxygenation impacts on proliferation of cancer cells and their sensitivity towards radio- and chemotherapy. Under low oxygen, mammalian cells show an adaptive response that leads to the induction of a number of genes with well-defined roles in oxygen supply and energy maintenance, e.g. genes encoding enzymes of the glycolytic pathway. The hypoxia-inducible factor 1 (HIF-1), a transcription factor consisting of the two proteins HIF-1alpha and HIF-1beta, plays a major role in the pleiotropic response observed under low oxygen. We have determined, by Northern analysis, the mRNA levels of HIF-1alpha and of two glycolytic enzymes known to be transcriptionally activated by HIF-1, namely phosphoglycerate kinase 1 (PGK 1) and pyruvate kinase M2 (PKM2), in different hepatoma cell lines and in mouse and human tissues. Hypoxic treatment of various mouse and human hepatoma cell lines led to the expected increase in the amount of PGK1 and PKM2 mRNA, while HIF-1alpha mRNA levels were not significantly elevated. Analysis of mouse liver tumors demonstrated no tumor-specific increases in HIF-1alpha or PGK1 mRNA levels. In five of eight human colorectal cancers investigated, PGK1 and PKM2 mRNA levels were increased in comparison to the corresponding normal tissues, while HIF-1alpha mRNA levels were not significantly changed. The majority of the colorectal cancers demonstrated p53 immunoreactivity, presumably due to mutation of the gene; there was, however, no correlation between the p53 staining pattern and mRNA expression levels of glycolytic enzymes.

The role of the tumor suppressor protein p53 in apoptosis of mouse hepatoma cells was studied. Different lines were used which were either p53 wild-type or carried various types of heterozygous or homozygous p53 mutations. The presence of mutations was demonstrated to correlate with a lack in transactivating activity of p53. While UV-light effectively produced apoptosis in cells of all lines, irrespective of their p53 mutational status, g-irradiation induced the formation of micronuclei but failed to induce apoptosis. Both UV-and girradiation led to nuclear accumulation and increases in p53 protein in p53 wild-type cells. Similarly, no significant differences in apoptotic response between p53 wild-type and p53 mutated cells were seen with other apoptotic stimuli like CD95/APO-1/Fas or TNFa. These data suggest that wildtype p53 is not required for induction of apoptosis in mouse hepatoma cells which may explain the apparent lack of p53 mutations in mouse liver tumors.