Keith Bonham

Transcription of the Human c-Src Promoter Is Dependent on Sp1, a Novel Pyrimidine Binding Factor SPy, and Can Be Inhibited by Triplex-forming Oligonucleotides

The tyrosine kinase pp60c-src has been implicated in the regulation of numerous normal physiological processes as well the development of several human cancers. However, the mechanisms regulating its expression have not been addressed. In the present study, we report the presence of two Sp1/Sp3 binding sites and three polypurine:polypyrimidine (Pu:Py) tracts in the c-Src promoter that are essential for controlling expression. We demonstrate that Sp1, but not Sp3, is capable of activating the c-Src promoter and that Sp3 is also capable of inhibiting Sp1-mediated transactivation. The presence of multiple Pu:Py tracts conferred S1 sensitivity on plasmids in vitro, suggesting they are capable of adopting non B-DNA conformations. These tracts specifically bind a nuclear factor we named SPy (Src pyrimidine binding factor), which demonstrates both novel double- and single-stranded binding specificities. Mutations eliminating SPy binding compromised Src transcriptional activity, especially in concert with additional mutations affecting Sp1 binding, suggesting the two factors may cooperate in regulating c-Src expression. Finally, we demonstrate that triplex-forming oligonucleotides designed to target both Sp1 and SPy binding sites can down-regulate c-Src expression in vitro, suggesting a potential therapeutic approach to controlling c-Src expression in diseases where aberrant expression or activity has been documented.

SRC transcriptional activation in a subset of human colon cancer cell lines.

Human SRC encodes the non‐receptor tyrosine kinase pp60c‐Src, which is activated in many human colon cancer cell lines (HCCLs) and tumors. We found that both c‐Src protein and mRNA levels were elevated in a subset of HCCLs. Increased c‐Src mRNA and protein levels correlated strongly with increased c‐Src kinase activity. Nuclear run‐on analysis and c‐Src mRNA half‐life determination demonstrated increased mRNA levels were due to increased transcription of the SRC gene. We also observed decreased c‐Src mRNA stability in cell lines that displayed SRC transcriptional activation. Our findings provide the first evidence that SRC transcriptional activation is an important determinant of c‐Src expression and activity in HCCLs.

Histone deacetylase inhibitors regulate p21WAF1 gene expression at the post-transcriptional level in HepG2 cells

Histone deacetylase inhibitors (HDIs) are thought to act primarily at the level of transcription inducing cell cycle arrest, differentiation and/or apoptosis in many cancer cell types. Induction of the potent cdk/cyclin inhibitor p21WAF1 is a key feature of this HDI mediated transcriptional re‐programming phenomenon. However, in the current study we report that HDIs are also capable of inducing p21WAF1 through purely post‐transcriptional events, namely increased mRNA stability. These studies highlight our growing appreciation for the complexities of HDI mediated effects and challenge our preconceptions regarding the action of these promising anti‐neoplastics.

The ubiquitous and tissue specific promoters of the human SRC gene are repressed by inhibitors of histone deacetylases.

Histone deacetylase inhibitors have generated keen interest as potential chemopreventive and chemotherapeutic agents due to their ability to induce cell cycle arrest, differentiation, and apoptosis in a diverse group of cancer derived cell lines. Activation of the 60 kDa non-receptor tyrosine kinase, c-Src, has been a consistent finding in many tumors and tumor derived cell lines, and has been implicated in these same cellular processes. We have shown that the histone deacetylase inhibitors, sodium butyrate and Trichostatin A, repressed c-Src mRNA and protein expression in a dose-dependent manner in cell lines derived from cancers of the colon, breast and liver. Our group has previously identified two distinct promoters that are responsible for SRC transcription, separated by a distance of approximately 1 kb. Sodium butyrate and Trichostatin A strongly inhibited activity of each of these highly disparate SRC promoters, demonstrating histone deacetylase inhibitors directly repress SRC transcription. This repression did not require protein neosynthesis and was not associated with a decrease in binding of protein factors essential for either promoters activity. Our finding that sodium butyrate and Trichostatin A inhibit both SRC promoters suggest this oncogene may be a major target of these agents, and may explain in part their anti-cancer activity.

SRC Proximal and Core Promoter Elements Dictate TAF1 Dependence and Transcriptional Repression by Histone Deacetylase Inhibitors

ABSTRACT Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation, or apoptosis in numerous cancer cell types both in vivo and in vitro. These dramatic effects are the result of a specific reprogramming of gene expression. However, the mechanism by which these agents activate the transcription of some genes, such as p21 WAF1 , but repress others, such as cyclin D1, is currently unknown. We have been studying the human SRC gene as a model for HDI-mediated transcriptional repression. We found previously that both the tissue-specific and housekeeping SRC promoters were equally repressed by HDIs. Here we show that, despite an overt dissimilarity, both SRC promoters do share similar core promoter elements and transcription is TAF1 dependent. Detailed analysis of the SRC promoters suggested that both core and proximal promoter elements were responsible for HDI-mediated repression. This was confirmed in a series of promoter-swapping experiments with the HDI-inducible, TAF1-independent p21 WAF1 promoter. Remarkably, all the SRC-p21 WAF1 chimeric promoter constructs were not only repressed by HDIs but also dependent on TAF1. Together these experiments suggest that the overall promoter architecture, rather than discrete response elements, is responsible for HDI-mediated repression, and they implicate core promoter elements in particular as potential mediators of this response.

N-Myristoyltransferase 2 expression in human colon cancer: Cross-talk between the calpain and caspase system

A number of viral and eukaryotic proteins which undergo a lipophilic modification by the enzyme N‐myristoyltransferase (NMT: NMT1 and NMT2) are required for signal transduction and regulatory functions. To investigate whether NMT2 contributes to the pathogenesis of colorectal carcinoma, we observed a higher expression of NMT2 in most of the cases of cancerous tissues compared to normal tissues (84.6% of cases; P < 0.05) by Western blot analysis. Furthermore, protein–protein interaction of NMTs revealed that m‐calpain interacts with NMT1 while caspase‐3 interacts with NMT2. Our findings provide the first evidence of higher expression of NMT2 in human colorectal adenocarcinomas and the interaction of both forms of NMT with various signaling molecules.

Requirement of N-Myristoyltransferase 1 in the Development of Monocytic Lineage

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/–) Nmt1-deficient mice. Homozygous (−/−) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c−Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.

Histone acetylation is not an accurate predictor of gene expression following treatment with histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors (HDIs) are documented for their role in activation and/or repression of gene expression. Currently, it is believed that HDAC inhibitors act at the histone level to alter chromatin dynamics through the inactivation of HDACs thereby resulting in histone hyperacetylation and increased transcriptional activation. However, transcriptional repression of gene expression is not explained by this model. Indeed, changes in the acetylation status of discreet lysine residues of histones associated with genes repressed by HDAC inhibitors have not been reported. Therefore, we carried out a systematic investigation of the changes in histone acetylation status at the promoter regions of two genes differentially affected by HDIs to gain a better understanding of how changes in histone acetylation correspond to changes in transcriptional activity.

CREB3L1 is a metastasis suppressor that represses expression of genes regulating metastasis, invasion, and angiogenesis.

ABSTRACT The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.

Src family kinase members have a common response to histone deacetylase inhibitors in human colon cancer cells

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation and/or apoptosis in numerous cancer cell types and have shown promise in clinical trials. These agents are particularly novel, given their ability to selectively influence gene expression. Previously, we demonstrated that the HDIs butyrate and trichostatin A (TSA) directly repress c‐Src proto‐oncogene expression in many cancer cell lines. Activation and/or overexpression of c‐Src have been frequently observed in numerous malignancies, especially of the colon. Therefore, our observation was particularly interesting since butyrate is a naturally abundant component of the large intestine and has been suggested to be a cancer‐preventive agent. However, c‐Src is not the only Src family kinase (SFK) member to be implicated in the development of human cancers, including those of the colon. Therefore, the relative expression levels of known SFKs were examined in a panel of human colon cancer cell lines. We found a surprisingly diverse expression pattern but noted that most cell lines expressed relatively high levels of at least 2 SFKs. When the effects of butyrate and TSA were examined in representative cell lines, the expression of all SFKs was repressed in a dose‐ and time‐dependent manner. Further, detailed examination of Lck, Yes and Lyn demonstrated that this repression had a direct effect on transcription and was independent of new protein synthesis. These results mirror our earlier data obtained with c‐Src and suggest that SFKs are a major target of HDIs and likely account in part for the anticancer effects of these promising new drugs.