Histone deacetylases and cancer.

Histone deacetylases (HDACs) regulate the expression and activity of numerous proteins involved in both cancer initiation and cancer progression. By removal of acetyl groups from histones, HDACs create a non-permissive chromatin conformation that prevents the transcription of genes that encode proteins involved in tumorigenesis. In addition to histones, HDACs bind to and deacetylate a variety of other protein targets including transcription factors and other abundant cellular proteins implicated in control of cell growth, differentiation and apoptosis. This review provides a comprehensive examination of the transcriptional and post-translational mechanisms by which HDACs alter the expression and function of cancer-associated proteins and examines the general impact of HDAC activity in cancer.

Retinoic acid- and bone morphogenetic protein 4-induced apoptosis in P19 embryonal carcinoma cells requires p27.

During development, many cells are specifically eliminated. Therefore, programmed cell death must be understood to fully elucidate embryogenesis. Retinoic acid (RA) and bone morphogenetic protein (BMP) 4 induce rapidly dividing P19 embryonal carcinoma cells to undergo apoptosis. RA alone minimally induces apoptosis, while BMP4 alone induces none. RA and BMP4 exposure also elevates the number of cells in the G1 phase of the cell cycle. Because many cell cycle proteins control both proliferation and apoptosis, we determined the role of these proteins in inducing apoptosis. Although the mRNA levels of cyclins D1 and D2 are reduced in cells undergoing apoptosis, the protein levels are not. In contrast, RA and BMP4 induce the Cdk inhibitor p27. This protein binds Cdk4 in RA- and BMP4-treated cells and inhibits Cdk4-dependent kinase activity. We used p27 antisense oligonucleotides to rescue the P19 cells from RA and BMP4 apoptosis thus proving that p27 is necessary. The Cdk4 substrate, retinoblastoma (Rb) protein, is also induced in apoptotic cells. Consistent with the decreased kinase activity of the apoptotic cells, this Rb protein is hypophosphorylated and presumably active. These data support the hypothesis that RA and BMP4 together induce the p27 protein leading to Rb activation and ultimately apoptosis.

The expression and activity of D-type cyclins in F9 embryonal carcinoma cells: modulation of growth by RXR-selective retinoids.

The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.

BMP4- and RA-induced apoptosis is mediated through the activation of retinoic acid receptor alpha and gamma in P19 embryonal carcinoma cells.

Some growth factors, for example, members of the transforming growth factor-beta family, can induce apoptosis in a variety of cells. Retinoic acid (RA) also causes apoptosis in several malignant cell types. We have previously demonstrated that, although BMP2 or BMP4 cannot induce apoptosis alone, BMP2 or BMP4 and RA synergize to induce apoptosis in 95% of P19 embryonal carcinoma cells within 4 days of treatment. Such treatment also prevents neuronal differentiation of these cells. Retinoids exert their many effects through any of six distinct nuclear receptors. These retinoid-activated transcription factors directly regulate genes involved in cellular response such as apoptosis. Complete understanding of how BMP and RA specifically induce cell death requires identification of the retinoid receptors controlling apoptosis. By using receptor-selective retinoid agonists and antagonists, we have obtained evidence suggesting that activation of RAR alpha or gamma is sufficient to induce apoptosis in BMP4-treated cells.

Induction of altered gene expression in early embryos.

This review focuses on known genes whose expression may be perturbed by teratogens during early embryogenesis (preorganogenesis). Teratogens may disrupt embryogenesis by modifying positional information. Genes controlling positional information include those specifying the primary body axes: anterior-posterior, dorsal-ventral, or left-right. These genes often encode transcription factors, whose regulation or activation can stimulate aberrant tissue differentiation and morphogenesis. Alternatively, teratogens may directly affect cell differentiation, proliferation, or apoptosis. Hydrophilic signalling molecules such as growth factors and hydrophobic molecules such as retinoids regulate these processes. The signalling pathways activated often induce the coordinate regulation of tissue specific gene expression. In addition to modifying individual signalling pathways, teratogens can synergize with or antagonize the effects of other teratogens through inappropriate interactions between signal transduction pathways. Since teratogens may often directly or indirectly perturb the expression of known or as yet undescribed developmentally critical genes, this review also provides a short description of techniques to identify genes whose expression is altered by teratogens.

Specific induction of apoptosis in P19 embryonal carcinoma cells by retinoic acid and BMP2 or BMP4.

Retinoic acid (RA) affects the response of many cells to growth factors, including the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-beta, family of growth factors, originally identified by their bone-inducing activities. Their widespread expression suggests many roles other than that in osteogenesis. Because RA modulates the cell's response to growth factors, this may be a means by which the retinoids exert some of their known teratogenic effects. One such cellular response may be apoptosis. While apoptosis is required for normal development, the location and timing of its induction must be carefully controlled. Recently, several TGF-beta family members have been implicated in the induction of apoptosis in certain cell types. We show here, using P19 embryonal carcinoma cells, that the combination of RA and BMP2 or BMP4 synergistically induces apoptosis in 40% of the population within 24 hr. In contrast, RA alone induces apoptosis in only 10-15% of the population and each of the BMPs alone minimally induces apoptosis. Apoptosis depends on the dose of both the RA and the BMP as well as on new protein synthesis. Further, the induction of apoptosis prevents the formation of fully differentiated neurons and glial cells and instead leads to primarily smooth muscle cell differentiation. These results suggest that some of the malformations caused by retinoids may be due to the induction of inappropriate apoptosis in cells exposed to BMPs.

The human lambda immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements.

We have recently reported the localization of the first transcriptional enhancer in the human lambda (lambda) immunoglobulin light chain locus. Enhancer activity was contained on a 1.2 kb SstI fragment, with partial activity retained on a core 111 bp PstI-SstI fragment. This enhancer is located 11.7 kb downstream of C lambda 7, the most 3' lambda constant region gene. Using a chloramphenicol acetyl transferase (CAT) assay system, we have now determined the boundaries of the complete enhancer and find it is two- to four-fold as active as the core fragment in both pre-B and B cell lines. Interestingly, a larger fragment, containing the complete enhancer as well as 5' and 3' flanking sequences has four- to eight-fold reduced activity when tested in pre-B cell lines, but full activity in B cell lines. This suggests the presence of developmentally regulated negative elements flanking the human lambda enhancer which prevent or reduce its activity at a developmentally incorrect time. By using in vivo footprinting we have begun to examine the protein interactions within this enhancer in a more physiologically relevant manner and have identified motifs which are shared with the murine lambda enhancers, as well as motifs unique to the human lambda enhancer.

Identification and localization of a developmental stage-specific promoter activity from the murine lambda 5 gene.

The lambda 5 protein is expressed in pre-B cells in association with VpreB and mu-heavy chains, and is critical for differentiation to the B cell stage. Pre-B cell-specific expression of the lambda 5 and VpreB genes is regulated at the level of transcription initiation. In this report, we have identified several DNase l-hypersensitive sites 2.5- to 6.0-kb downstream of the lambda 5 gene, which are present in the pre-B cell line 70Z/3, but not in the myeloma cell line j558L. These sites, however, were shown to have no transcriptional enhancer activity as measured by transient transfection. Enhancer activity was identified within a 361-bp fragment (-296 to +65, where +1 is the major 5' transcription initiation site) upstream of the mouse lambda 5 gene. This activity is orientation and position independent, and is also tissue and differentiation stage specific (active in pre-B but not B and T cells). Deletion constructs indicate that three adjacent areas (-210 to -169, -153 to -64, and -64 to -22) are all necessary for enhancer activity. Pre-B cell-specific promoter activity was shown to reside within the -219 to +109 fragment. Basal promoter activity resides within the -64 to +109 fragment, but is not tissue specific or stage specific. A negative element within the -101 to -64 region is active in all lymphoid cell lines tested and therefore cannot by itself be responsible for the tissue and stage specificity. The data indicate that the elements responsible for the enhancer activity (-210 to -22) are part of the lambda 5 gene promoter and likely confer the tissue and stage specificity via positive elements within the -210 to -22 region.