The deoxycytidine analog 5-aza-2'-deoxycytidine (5-azadCyd) has been widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation. Prior to the availability of mutant mice with altered DNA methyltransferase levels, treatment of cells with drugs has been the only means to experimentally manipulate the level of genomic DNA methylation in mammalian cells. Substitution ofDNA with 5-azadCyd leads to covalent trapping of the enzyme, thereby depleting the cells of enzyme activity and resulting in DNA demethylation. 5-AzadCyd or 5-azacytidine treatment causes multiple changes in treated cells, including activation of silent genes, decondensation of chromatin, and induction ofcellular differentiation, all of which are believed to be consequences of drug-induced demethylation. 5-AzadCyd is highly toxic in cultured cells and animals and is utilized as a potent antitumor agent for treatment of certain human cancers. It has been postulated that the toxicity of the drug in mammalian cells is also due to its inhibition of DNA methylation. The chemistry of the methylation reaction is consistent, however, with an alternative mechanism: the cytotoxic effect of 5-azadCyd may be directly mediated through the covalent binding of DNA methyltransferase to 5-azadCydsubstituted DNA. We have tested this possibility by using embryonic stem cells and mice with reduced levels of DNA methyltransferase due to a targeted mutation ofthe gene. When exposed to 5-azadCyd mutant embryonic stem cells or embryos were sigfntly more resistant to the toxic effects of the drug than wild-type cells and embryos, respectively. These results strongly suggest that the cellular DNA methyltransferase itself, rather than the secondary demethylation of genomic DNA, is the primary mediator of5-azadCyd cytotoxicity. In light ofour results, some conclusions from previous studies using 5-azadCyd in order to experimentally manipulate cellular methylation levels may have to be reassessed. Also, our data make clear predictions for cancer treatment: tumor cells with elevated DNA methyltransferase levels would be expected to be susceptible to treatment with 5-azadCyd, whereas tumors with re-

Matrix metalloproteinases (MMPs) are synthesized as latent proenzymes. A proteolytic cleavage event involving processing of the cysteine-rich N-terminal propeptide is required for their full activation. Previous in vitro studies indicated that activation of proMMP-2 can occur through formation of a trimolecular complex between MMP-14, TIMP-2, and proMMP-2 at the cell surface. Using TIMP-2-deficient mice and cells derived from them, TIMP-2 was shown to be required for efficient proMMP-2 activation both in vivo and in vitro. The requirement for TIMP-2 was not cell-autonomous as exogenously added TIMP-2 could restore activation of proMMP-2 to TIMP-2-deficient cells. Mutant mice were overtly normal, viable, and fertile on the C57BL/6 background, indicating that both TIMP-2 and activated proMMP-2 are dispensable for normal development.More than 20 matrix metalloproteinases (MMPs) 1 have been described that collectively degrade all extracellular matrix and a number of non-matrix proteins involved in inflammation and cell growth control (reviewed in Ref. 1). The MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) have been associated directly and indirectly with many developmental processes such as branching morphogenesis (2-6), regulation of cell migration (7), apoptosis (8, 9), angiogenesis (10), and regulation of innate immunity (11).2 In addition to their link with developmental events, MMPs have been implicated in several disease processes such as tumor metastasis (12), arthritis (13), and emphysema (14,15). They are regulated in many ways that include transcriptional (16) and post-translational mechanisms. Two post-translational means of MMP regulation have been described. First, MMPs are synthesized as latent pro-enzymes. A cysteine-rich N-terminal peptide of the latent proenzyme interacts with the Zn ϩ2 at the active site, blocking proteolytic activity of the proteinase (17). After cleavage of the propeptide, the latent enzyme becomes activated, enabling it to degrade its substrate. A second mode of post-translational regulation has been described as well. Active MMPs are inhibited by the broad spectrum proteinase inhibitor ␣ 2 -macroglobulin (18) and by four known MMP-specific tissue inhibitors of metalloproteinases or TIMPs (19 -22). The mechanisms by which MMPs are proteolytically processed from their latent to their activated forms and how they are regulated by inhibitors are important to understanding their contributions to normal developmental and pathological processes.Evidence exists for several mechanisms of proteolytic activation of the latent proMMPs. For example, plasmin can participate in the activation of proMMP-1(23), -3 (24), -7 (25), -9 (26, 27), -13 (28), and -14 (29), whereas furin can activate the membrane-associated proMMP-14 (30), proMMP-11 (31), and others. Already activated MMPs can also contribute to activation of other proMMPs. For example, MMP-3 can activate proMMP-1 (32), and -proMMP-7 (25) and MMP-14 together with MMP-2 can contribute to the activ...

It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

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Sequence-specific methylation of the promoter and adjacent regions in mammalian genes transcribed by RNA polymerase II leads to the inhibition of these genes. So far, RNA polymerase ITT-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase ITTtranscribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by Hpall (5'-CCGG-3') and Hhal (5'-GCGC-3'). Three of these 5'-CG-3' sequences are located close to the internal regulatory region of the VAI segment. An unmethylated, a 5'-CCGG-3'and 5'-GCGC-3'-methylated, and a 5'-CG-3'-methylated pUC18 construct containing the VAI and VAII regions were transfected into mammalian cells. In many experiments, an inactivating effect of 5'-CCGG-3' and 5'-GC GC-3' DNA methylation on the VAI region was not observed. In contrast, methylation of all 20 5'-CG-3' sequences in the VAI region by a CpG-specific DNA methyltransferase from Spiroplasma species did interfere with VAI transcription. Transcription of the VAIand VAIIand of the VAI-containing constructs was also shown to be inhibited in an in vitro cell-free transcription system after the constructs had been methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences or at all 5'-CG-3' sequences. When an oligodeoxyribonucleotide which carried the internal control block A of the VAI region was methylated at three 5'-CG-3' sequences, the formation of a complex with HeLa nuclear proteins was abrogated. The results presented support the notion that the VAI gene transcribed by the DNA-dependent RNA polymerase III is also inactivated by methylation of the decisive 5'-CG-3' sequences.