We have shown previously that the heavy metalresponsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1 -/-embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the γ-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-κB (p65/RelA).

Telomere shortening impairs proliferation of transformed cells but also leads to cancer initiation by inducing chromosomal instability. Here, we discuss recent developments in our understanding of the role of telomeres in replication stress and how telomerase expression in somatic stem cells may affect genome integrity control and carcinogenesis.

Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H 2 O 2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H 2 O 2 ; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.All living organisms are able to cope with a variety of stress situations by immediately adapting their gene expression program to the stress stimulus. For example, metallothioneins, small cysteine-rich proteins, are strongly upregulated upon heavy metal load (2,27,29,49,63,70,71). They have the ability to bind and hence neutralize toxic (such as Cd and Hg) and excess nontoxic (such as Zn and Cu) heavy metal ions and also act as radical scavengers. Metal response element binding transcription factor 1, also called metal-responsive transcription factor 1 (MTF-1), plays an important role in the cellular response to heavy metal stress (2,21,38,55,75) and is essential for embryonic liver development in the mouse (24). MTF-1 contains six zinc fingers of the C 2 H 2 type. C-terminal to the zinc fingers are three distinct activation domains, an acidic, a proline-rich, and a serine/threonine-rich domain. Via the zinc fingers, it binds to DNA sequence motifs with the consensus binding site TGCRCNC, known as metal response elements (MREs). MREs are present in the promoters of metallothionein genes (41,61,64) and other target genes, including zinc transporter ZnT1 and ␥-glutamylcysteine synthetase heavy chain (24,34,37). MTF-1 is also involved in the responses to oxidative stress (16, 24), hypoxia (23, 46), and amino acid starvation (1). In quiescent cells, MTF-1 pref...

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The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy-controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb-dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase-deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy-induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.

Telomerase activity is repressed in most human somatic tissues during differentiation processes but strongly upregulated in most human tumors. Regulation of human telomerase activity primarily occurs at the level of transcriptional initiation of the TERT gene, which encodes the catalytic subunit of telomerase. We have generated a novel transgenic mouse model to study the regulation of the human TERT gene promoter in an in vivo system. For this purpose, we have cloned an 8.0-kbp human TERT promoter fragment in front of the bacterial lacZ reporter gene (hTERTp-lacZ), which encodes the B-galactosidase enzyme. Expression of the reporter gene was monitored by reverse transcription-PCR analysis, 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside staining of whole mount preparations, and histologic sections. We find that the activity of the human TERT promoter in most normal mouse tissues recapitulates the expression of the hTERT gene in normal human tissues and is under tighter control when compared with the endogenous mouse TERT gene expression. In testis, where highest lacZ expression was observed, the expression of the reporter gene was restricted to the spermatogonial stem cells and the spermatocytes. Intriguingly, we find increased levels of lacZ expression in mammary tumors of hTERTp-lacZ Â p53 +/À bitransgenic mouse mammary tumor model. Thus, this transgenic mouse model provides a suitable in vivo system to analyze the expression of the human TERT gene under physiologic conditions and during tumorigenesis. (Cancer Res 2005; 65(4): 1187-96)

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