Molecular events associated with trisomy of chromosome 7 in mouse skin chemical carcinogenesis

The primary objective of this study has been to investigate the effects at the molecular level of trisomy of mouse chromosome 7 in chemically induced skin tumors. It was previously proposed that the initiation event in the mouse skin carcinogenesis model is a heterozygous mutation of the Ha-ras-1 gene, mapped to chromosome 7. Previous studies in this laboratory identified trisomy 7 as one of the primary nonrandom cytogenetic abnormalities found in the majority of severely dysplastic papillomas and squamous cell carcinomas induced in SENCAR mice by an initiation-promotion protocol. Therefore, the first hypothesis tested was that trisomy 7 occurs by specific duplication of the chromosome carrying a mutated Ha-ras-1 allele. Results of a quantitative analysis of normal/mutated allelic ratios of the Ha-ras-1 gene confirmed this hypothesis, showing that most of the tumors exhibited overrepresentation of the mutated allele in the form of 1/2, 0/3, and 0/2 (normal/mutated) ratios. In addition, histopathological analysis of the tumors showed an apparent association between the degree of malignancy and the dosage of the mutated Ha-ras-1 allele. To determine the mechanism for loss of the normal Ha-ras-1 allele, found in 30% of the tumors, a comparison of constitutional and tumor genotypes was performed at different informative loci of chromosome 7. By combining Southern blot and polymerase chain reaction fragment length polymorphism analyses of DNAs extracted from squamous cell carcinomas, complete loss of heterozygosity was detected in 15 of 20 tumors at the Hbb locus, and in 5 of 5 tumors at the int-2 locus, both distal to Ha-ras-1. In addition, polymerase chain reaction analysis of DNA extracted from papillomas indicated that loss of heterozygosity occurs in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion, suggesting that this event may be associated to the acquisition of the malignant phenotype. Allelic dosage analysis of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1, indicated that loss of heterozygosity on mouse chromosome 7 occurs by a mitotic recombination mechanism. Overall, these findings suggest the presence of a putative tumor suppressor locus on the 7F1-ter region of mouse chromosome 7. Thus, loss of function by homozygosis at this putative suppressor locus may complement activation of the Ha-ras-1 gene during tumor progression, and might be associated with the malignant conversion stage of mouse skin carcinogenesis. ^

Molecular analysis of the human $\gamma$-chain T cell receptor genes

In our studies we have focused on the issue of variability and diversity of the $\gamma$ (or $\delta)$ chain T cell receptor (TCR) genes by studying cDNA transcripts in peripheral blood mononuclear cells or $\gamma\delta$ TCR+ T cell clones. The significance of these studies lies in the better understanding of the molecular biology of the $\gamma\delta$ T cell receptor as well as in answering the question whether certain molecular forms predominate in $\gamma\delta$ T cells exhibiting specific immunologic functions. We establish that certain $\gamma$-chain TCR genes exhibit particular patterns of rearrangements in cDNA transcripts in normal individuals. V$\gamma$I subgroup were shown to preferentially rearrange to J$\gamma$2C$\gamma$2 gene segments. These preferential VJC rearrangements, may have implications regarding the potential for diversity and polymorphism of the $\gamma$-chain TCR gene. In addition, the preferential association of V$\gamma$I genes with J$\gamma$2C$\gamma$2, which encode a non-disulfide-linked $\gamma\delta$ TCR, suggests that $\gamma$ chains utilizing V$\gamma$I are predominantly expressed as non-disulfide-linked $\gamma\delta$ TCR heterodimers. The implications of this type of expression remain to be determined. We identified two alternative splicing events of the $\gamma$-chain TCR genes occurring in high frequency in all the normal individuals examined. These events may suggest additional mechanisms of regulation and control as well as diversification of $\gamma\delta$ TCR gene expression. The question whether particular forms of $\gamma$ or $\delta$-chain TCR genes are involved in HLA Class I recognition by specific $\gamma\delta$ cytotoxic T cell clones was addressed. Our results indicated that the T cell clones expressed identical $\gamma$ but distinct $\delta$-chains suggesting that the specificity for recognition of HLA-A2 or HLA-A3 may be conferred by the $\delta$-chain TCR. The issue of the degree of diversity and polymorphism of the $\delta$-chain TCR genes in a patient with a primary immunodeficiency (Omenn's syndrome) was addressed. A limited pattern of rearrangements in peripheral blood transcripts was found, suggesting that a limited $\gamma\delta$ TCR repertoire may be expressed in this particular primary immunodeficiency syndrome. Overall, our findings suggest that $\delta$-chain TCR genes exhibit the potential for significant diversity and that there are certain preferential patterns of expression that may be associated with particular immunologic functions. ^

Nuclear functions of dynein light chain 1 in hormone action

It is widely accepted that the process of breast cancer tumorigenesis involves estrogen receptor-alpha (ER)-regulated stimulatory pathways, which feed into survival, cell cycle progression and proliferative response. Recent data from Kumar laboratory indicate that dynein light chain 1 (DLC1) plays a role in survival, motility and invasiveness, all of which are required for a successful tumorigenesis process. In the present research, we have discovered a mechanistic bidirectional regulatory link between the DLC1 and ER. We found that DLC1 facilitates ligand-induced ER transactivation involving the recruitment of the DLC1-ER complex to ER-target genes. To gain insights into the mechanism by which DLC1 regulates the ER pathway, we set out to identify novel DLC1-interacting proteins. Among other proteins, we identified KIBRA and Ciz1 as two novel DLC1-interacting proteins. We found that the KIBRA-DLC1 complex is recruited to ER-responsive promoters, and that KIBRA-DLC1 interaction is needed for the recruitment of ER to its targets as well as for ER's transactivation function. Finally, we found that KIBRA utilizes its histone H3interacting glutamic acid-rich region to regulate the transactivation activity of ER. During the course of this work, we also discovered that DLC1 interacts with Cdk2 and Ciz1, and such interactions play a direct accelerating role in the G1-S transition of breast cancer cells. While delineating the role of Ciz1 in hormone-responsive cancer cells, we found that Ciz1 is an estrogen-responsive gene, and acts as a co-regulator of ER. Accordingly, Ciz1 overexpression in breast cancer cells conferred estrogen hypersensitivity, promoted the growth-rate, anchorage-independency and tumorigenic properties. Collectively, findings made during the course of the present dissertation research introduced two new molecular players in the action of ER in breast cancer cells, with a particular focus on cell cycle progression and ER-chromatin target regulation. In addition, findings presented here provide novel mechanistic insight about the contribution of DLC1 and its interacting proteins in amplifying the hormone action and promoting the process of breast cancer tumorigenesis. ^

Ser88 phosphorylation regulates dynein light chain 1 (DLC1) function in the mouse mammary gland

Dynein light chain 1 (DLC1) is a highly conserved and ubiquitously expressed protein which might have critical cellular function as total loss of DLC1 caused Drosophila embryonic death. Despite many proteins and RNAs interaction with it identified, DLC1's function(s) and regulation are largely unknown. Recently, DLC1 was identified as a physiological substrate of P21-activate kinase 1(Pak1) kinase from a human mammary cDNA library in a yeast-2-hybridization screening assay. Studies in primary human tumors and cell culture implicated that DLC1 could promote mammary cancerous phenotypes, and more importantly, Ser88 phosphorylation of DLC1by Pak1 kinase was found to be essential for DLC1's tumorigenic activities. Based on the above tissue culture studies, we hypothesized that Ser88 phosphorylation regulates DLC1. ^ To test this hypothesis, we generated two transgenic mouse models: MMTV-DLC1 and MMTV-DLC1-S88A mice with mammary specific expression of the DLC1 and DLC1-S88A cDNAs. Both of the transgenic mice mammary glands showed rare tumor incidence which indicated DLC1 alone may not be sufficient for tumorigenesis in vivo. However, these mice showed a significant alteration of mammary development. Mammary glands from the MMTV-DLC1 mice had hyperbranching and alveolar hyperplasia, with elevated cell proliferation. Intriguingly, these phenotypes were not seen in the mammary glands from the MMTV-S88A mice. Furthermore, while MMTV-DLC1 glands were normal during involution, MMTV-S88A mice showed accelerated mammary involution with increase apoptosis and altered expression of involution-associated genes. Further analysis of the MMTV-S88A glands showed they had increased steady state level of Bim protein which might be responsible for the early involution. Finally, our in vitro data showed that Ser88 phosphorylation abolished DLC1 dimer and consequently might disturb its interaction with Bim and destabilize Bim. ^ Collectively, our findings provided in vivo evidence that Ser88 phosphorylation of DLC1 can regulate DLC1's function. In addition, Ser88 phosphorylation might be critical for DLC1 dimer-monomer transition. ^