A large percentage of human hepatomas produce a-fetoprotein (AFP), but the levels of AFP expression vary greatly among hepatomas. To understand the molecular basis for this variation, we analyzed tanscriptional regulatory activities associated with the 5'-flanking region of the AFP gene in two human hepatoma cell lines, HuH-7 and huH-1/cl-2, which produce a high and a low level of AFP, respectively. We found that the low level of AFP production in huH-1/cl-2 is due to the action of at least two silencer regions located between the enhancer and the promoter of the AFP gene. In contrast, no silencer activity is expressed in HuH-7. We identified 5'-CTTCATAACTAATACTT-3' to be a core sequence responsible for the negative regulatory activity. This sequence is repeated four times in a strong, distal silencer region, Sd, whereas one copy is present in a weak, proximal silencer region, Sp. The silencer reduces transcriptional initiation by blocking enhancer activation of the AFP promoter in a position-dependent manner. The silencer functions in the presence of positive transcription factors and may play a key role in developmental repression as well as variable expression of the AFP gene in hepatomas.The a-fetoprotein (AFP) gene is a developmentally regulated gene whose activity decreases rapidly after birth and becomes hardly detectable in adult life. However, the AFP gene is often reactivated to a high level in hepatocellular carcinoma (HCC) and teratocarcinomas (1, 4, 36). In contrast, the expression of the albumin gene, which is evolutionarily related to the AFP gene, steadily increases during normal development and stays high throughout adult life.Elevated levels of AFP have been observed in more than 70% of HCC patients, making AFP as a reliable diagnostic and prognostic marker of HCC. The AFP levels among AFP-positive patients, however, vary widely. Analysis of mechanisms underlying the variation of AFP production in HCC is of importance from clinical as well as basic biological standpoints.HuH-7 and huH-1/cl-2 are two human hepatoma cell lines that exhibit various liver-specific functions (23-25). HuH-7 produces a large amount of AFP and a moderate level of albumin. huH-1/cl-2, on the other hand, produces only a detectable amount of AFP but a high level of albumin. These cell lines provide useful model systems for the study of the molecular basis for different levels of expression of the AFP and albumin genes in hepatoma.We have shown previously that in HuH-7 cells, AFP gene expression is regulated positively by the enhancer, promoter, and glucocorticoid-responsive element (27,32,39). We have not so far detected DNA elements that suppress AFP gene transcription in this cell line. In this study, we analyzed mechanisms responsible for a low level of AFP gene expression in huH-1/cl-2. Two possibilities were considered: (i) the reduced activity of positive regulatory elements and (ii) the activation of negative control elements. We present evidence that the latter mechanism is largely * Corresponding author. t Presen...

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Apolipoprotein Al (Apo Al) is the major protein component of high density lipoprotein (HDL) particles.HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo Al will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo Al promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to Identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo Al gene.

a-Fetoprotein (AFP) is one of the oncofetal proteins.Received October 20, 1995; accepted January 10, 1996. An elevated level of serum AFP is known to be ob- cific enhancers exist in a far upstream region (04.0-

Alpha-fetoprotein (AFP) producing gastric cancer (AFP ± GC) is very malignant and highly metastatic compared with common gastric cancer. However, the causal relationship between AFP production and the high malignancy of AFP-GC is unclear. We investigated AFP gene regulation in AFP-GC by an active transcription factor, HNF1 (hapatocyte nuclear factor 1) and a repressive transcription factor, ATBF1 (AT motif binding factor 1). RNase protection assays revealed that the production of AFP in gastric cancer cells did not directly associate with HNF1 expression. An inverse relation between the expressions of ATBF1 and AFP was clearly observed in gastric cancer cells. CAT assays showed the direct inhibition of AFP gene expression by ATBF1. Methylation analysis of the AFP promoter region in gastric cancer cells suggested that methylation itself could not explain the silencing of the AFP gene. Immunohistochemistry of resected clinical samples revealed that AFP producing cells lacked ATBF1 immunoreactivity. Our data suggests that the absence of ATBF1 is responsible for AFP gene expression in gastric cancer, and the absence of ATBF1 is a distinct characteristic of AFP-GC and might be important for its highly malignant nature. Oncogene (2001) 20, 869 ± 873.