Retrospective assesment of the levonorgestrel intrauterine device to treat complex atypical hyperplasia and early endometrioid endometrial cancer

Objective: The primary objective of this project was to describe the efficacy of the Levonorgestrel Intrauterine Device (LIUD) for treatment of Complex Endometrial Cancer (CAH) and Grade 1 Endometrial Cancer (G1EEC) in terms of rate of Complete Response (CR) and Partial Response (PR) after 6 months of therapy. Finally, we assessed if any clinical or pathologic features were associated with response to the LIUD. ^ Methods: This study was a retrospective case series designed to report the response rate of patients with CAH or G1EEC treated with LIUD therapy. In addition, this study has a laboratory component to assess molecular predictors of response to LIUD therapy. Retrospective data already collected from patients diagnosed with CAH or EEC grade 1 and treated with LIUD therapy at MD Anderson Cancer Center (MDACC) were used for this study. Patients from all ethnic and race groups were included. A Complete Response (CR) was defined in patients diagnosed with CAH if pathologic report at 6 months demonstrated either no evidence of hyperplasia or no atypia in the setting of simple or complex hyperplasia. Partial Response (PR) was recorded if disease downgraded to only CAH from G1EEC. No Response (NR) was recorded if pathologic report demonstrates no change (Stable Disease, SD) or progression to cancer (Progressive Disease, PD). We calculated the proportion of patients with complete response to LIUD therapy with 95% confidence interval. We compared the response rates (CR/PR vs NR) by obesity status (Obese if BMI > 40 kg/m2 vs non-obese if BMI <= 40 kg/m2) as well as other clinical and pathologic factors, such as age, uterine size (median size), and presence of exogenous progesterone effect. ^ Results: There were 39 patients diagnosed with either CAH or G1EEC treated with the LIUD. Of 39 patients, 12 did not have pathological results of biopsy at 6months time period. Of 27 evaluable patients, 17 were diagnosed with CAH and 10 with G1EEC. Overall response rate (RR) was 78% (95% CI = 62-94%) at 6 months, 18 patients had CR (4 in G1EEC; 14 in CAH), 3 patients had PR (3 in G1EEC), 3 had SD (1 in CAH; 2 in G1EEC), 3 had PD (2 in CAH; 1 in G1EEC). After histology stratification, RR at 6 months was 82.35% (14/17; 95%CI = 67.4-97.3%) in CAH and 70% (7/10; 95% CI = 41-98.4%) in G1EEC. ^ There was no difference in response (R) and no response (NR) based on BMI (p=0.56). He observed a trend showing association between age with response (p=0.1). There was no association between uterine size and response to therapy (p=0.17). We recorded strong association between exogenous progesterone effect and response. ^ Conclusion: LIUD therapy for the treatment of CAH and G1EEC may be effective and safe. Presence of exogenous progesterone effect may predict the response to LIUD therapy at earlier time points. There is need of further studies with larger sample size to explore the relationship of response with other clinical and pathologic factors^

THE p63 ISOFORM ∆Np63α INHIBITS EPITHELIAL – MESENCHYMAL TRANSITION BY PROMOTING THE EXPRESSION OF MIR-205 IN HUMAN BLADDER CANCER CELLS

p63, a p53 family member, is a transcription factor that has complex roles in cancer. This study focuses on the role of the ∆Np63α isoform in bladder cancer (BC). Epithelial – mesenchymal transition (EMT) is a physiological process that plays an important part in metastasis and drug resistance. At the molecular level, EMT is characterized by the loss of the epithelial marker E-cadherin, and the acquisition of the transcriptional repressors of E-cadherin (ZEB1, ZEB2, TWIST, SNAI1 and SNAI2). Recent publications highlight the role of microRNAs belonging to the miR-200 family and miR-205 in preventing EMT through suppression of ZEB1 and ZEB2. p53, the homologue of p63, is implicated in regulating EMT by modulating the expression of miR-200c; however, the mechanisms underlying miR-205 control remain unclear. Here we show that ∆Np63α regulates the transcription of miR-205 and controls EMT in human BC cells. We observed a strong correlation between the expression of ∆Np63α, miR-205 and E-cadherin in a panel of BC cell lines (n=28) and also in bladder primary tumors from a cohort of patients (n=98). A remarkably inverse correlation is observed between ∆Np63α and ZEB1/2 in cell lines. Stable knockdown (KD) ∆Np63α in UC6, an “epithelial” BC cell line, decreased the expression of miR-205 and induced ZEB1/2 expression, the effects that were reversed by expression of exogenous miR-205. Moreover, overexpressing ∆Np63α in UC3, a “messenchymal” BC cell line, brought about opposite results, an increase in miR-205 expression and a reduction in ZEB1/2 expression. Modulation of ∆Np63α expression resulted in a parallel change in the expression of miR-205 and miR-205 “host” gene (miR-205HG). Nuclear run-on and chromatin immunoprecipitation experiments demonstrated that ∆Np63α regulates the transcription of miR-205 through controlling the recruitment of RNA Polymerase II to the promoter of miR-205HG. Interestingly, high miR-205 expression correlated with poor clinical outcome in BC patients, consistent with our recent publication highlighting the enrichment of ∆Np63 in a lethal subset of muscle invasive BC. In summary, our data present the important roles of ∆Np63α in preventing EMT mediated by miR-205. Our study also identifies miR-205 as a potential molecular marker to predict clinical outcome in BC patients.

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

Scanned Ion Beam Therapy for Thoracic Tumors

Although frequently cured of Hodgkin lymphoma, adolescents and young adults can develop radiation induced second cancers. These patients could potentially benefit from scanned ion radiotherapy yet likely would require motion mitigation strategies. In theory, four-dimensional (4D) optimization of ion beam fields for individual motion states of respiration can enable superior sparing of healthy tissue near moving targets, compared to other motion mitigation strategies. Furthermore, carbon-ion therapy can sometimes provide greater relative biological effectiveness (RBE) for cell sterilization in a target but nearly equivalent RBE in tissue upstream of the target, compared to proton therapy. Thus, we expected that for some patients with Hodgkin lymphoma, carbon-ion therapy would reduce the predicted risk of second cancer incidence in the breast compared with proton therapy. The purpose of this work was to determine whether 4D-optimized carbon-ion therapy would significantly reduce the predicted risk of radiation induced second cancers in the breast for female Hodgkin lymphoma patients while preserving tumor control compared with proton therapy. To achieve our goals, we first investigated whether 4D-optimized carbon beam tracking could reduce dose to volumes outside a moving target compared with 3D-optimized carbon beam tracking while preserving target dose coverage. To understand the reliability of scanned carbon beam tracking, we studied the robustness of dose distributions in thoracic targets to uncertainties in patient motion. Finally, we investigated whether using carbon-ion therapy instead of proton therapy would significantly reduce the predicted risk of second cancer in the breast for a sample of Hodgkin lymphoma patients. We found that 4D-optimized ion beam tracking therapy can reduce the maximum dose to critical structures near a moving target by as much as 53%, compared to 3D-optimized ion beam tracking therapy. We validated these findings experimentally using a scanned carbon ion synchrotron and a motion phantom. We found scanned carbon beam tracking to be sensitive to a number of motion uncertainties, most notably phase delays in tracking, systematic spatial errors, and interfractional motion changes. Our findings indicate that a lower risk of second cancer in the breast might be expected for some Hodgkin lymphoma patients using carbon-ion therapy instead of proton therapy. For our reference scenario, we found the ratio of risk to be 0.77 ± 0.35 for radiogenic breast cancer after carbon-ion therapy versus proton therapy. Our findings were dependent on the RBE values for tumor induction and the radiosensitivity of breast tissue, as well as the physical dose distribution.

Expression and function of $\beta$-1,4-galactosyltransferase during wild-type and T/{\it t\/} -mutant spermatogenesis

In the mouse, gamete recognition is mediated in part by the binding of sperm surface $\beta$1,4 galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida ZP3. The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion. In this study, the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis was characterized and the role of GalTase was analyzed in transmission ratio distortion. It was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases and meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice.^ The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from +/t males. Results show that +- and t-spermatids have similar levels of both GalTase RNA and protein, indicating that transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.^ The presence of GalTase on the surface of an early spermatogenic cells before it is required on the mature sperm to perform its function during gamete binding suggests a separate function for GalTase in Sertoli-germ cell adhesion. Studies indicate that cell surface GalTase partly mediates the initial adhesion of pachytene spermatocytes, but not haploid spermatids, to Sertoli cells. ^

Isolation and characterization of cDNAs encoding novel homeoproteins from chondrocytes

Bone morphogenesis is a complex biological process. The multistep process of chondrogenesis is the most important aspect of endochondral bone formation. To study the mechanisms which control this multistep pathway of chondrogenesis during embryonic development, I started by isolating cDNAs encoding novel transcriptional factors from chondrocytes. Several such cDNAs encoding putative homeoproteins were identified from a rat chondrosarcoma cDNA preparation. I have been concentrating on characterizing two of these cDNAs. The deduced amino acid sequence of the first homeoprotein, Cart-1, contains a prd-type homeodomain. Northern hybridization and RNase protection analysis revealed that Cart-1 RNAs were present at high levels in a well differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 transcripts were also detected in primary chondrocytes, but not in numerous other cell types except very low levels in testis. In situ hybridization of rat embryos at different stages of development revealed relatively high levels of Cart-1 RNAs in prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. It is speculated that Cart-1 might play an important role in chondrogenesis. The second putative homeoprotein, rDlx, contains a Distal-less-like homeodomain. rDlx RNAs were also present at high levels in the rat chondrosarcoma tumor and in the cell line derived from this tumor. In situ hybridization of rat embryos revealed high levels of rDlx transcripts in the developing cartilages and perichondria of mature cartilages. rDlx transcripts were also detected in a number of nonchondrogenic tissues such as forebrain, otic vesicles, olfactory epithelia, apical ectodermal ridge (AER) of limb buds, the presumptive Auerbach ganglia of gastrointestinal tract. The unique expression pattern of rDlx suggests that it might play important roles in chondrogenesis and other aspects of embryogenesis. ^

Calcium-dependent mechanisms and long-term potentiation: Role of NMDA receptors, voltage -dependent calcium channels and persistent protein kinase activity

Long-term potentiation (LTP) is a rapidly induced and long lasting increase in synaptic strength and is the leading cellular model for learning and memory in the mammalian brain. LTP was first identified in the hippocampus, a structure implicated in memory formation. LTP induction is dependent on postsynaptic Ca2+ increases mediated by N-methyl-D-aspartate (NMDA) receptors. Activation of other postsynaptic routes of Ca2+ entry, such as voltage-dependent Ca2+ channels (VDCCs) have subsequently been shown to induce a long-lasting increase in synaptic strength. However, it is unknown if VDCC-induced LTP utilized similar cellular mechanisms as the classical NMDA receptor-dependent LTP and if these two forms of LTP display similar properties. This dissertation determines the similarities and differences in VDCC and NMDA receptor-dependent LTP in area CA1 of hippocampal slices and demonstrates that VDCCs and NMDA receptors activate similar cellular mechanisms, such as protein kinases, to induce LTP. However, VDCC and NMDA receptor activated LTP induction mechanisms are compartmentalized in the postsynaptic neuron, such that they do not interact. Consistent with activation properties of NMDA receptors and VDCCs, NMDA receptor and VDCC-dependent LTP have different induction properties. In contrast to NMDA-dependent LTP, VDCC-induced potentiation does not require evoked presynaptic stimulation or display input specificity. These results indicate that there are two different routes of postsynaptic Ca2+ which can induce LTP and the compartmentation of VDCCs and NMDA receptors and/or their resulting Ca2+ increases may account for the distinction between these LTP induction mechanisms.^ One of the molecular targets for postsynaptic Ca2+ that is required for the induction of LTP is protein kinases. Evidence for the role of protein kinase activity in LTP expression is either correlational or controversial. We have utilized a broad range and potent inhibitors of protein kinases to systematically examine the temporal requirement for protein kinases in the induction and expression of LTP. Our results indicate that there is a critical period of persistent protein kinase activity required for LTP induction activated by tetanic stimulation and extending until 20 min after HFS. In addition, our results suggest that protein kinase activity during and immediately after HFS is not sufficient for LTP induction. These results provide evidence for persistent and/or Ca2+ independent protein kinase activity involvement in LTP induction. ^

Functional analysis of p53 phosphorylation and a p53 hot spot mutation

The tumor suppressor p53 is a phosphoprotein which functions as a transcriptional activator. By monitoring the transcriptional activity, we studied how p53 functions is regulated in relation to cell growth and contact inhibition. When cells were arrested at G1 phase of the cell cycle by contact inhibition, we found that p53 transactivation function was suppressed. When contact inhibition was overridden by cyclin E overexpression which stimulates cell cycle progression, p53 function was restored. This observation led to the development of a cell density assay to study the regulation of p53 function during cell cycle for the functional significance of p53 phosphorylation. The murine p53 is phosphorylated at serines 7, 9, 12, 18, 37, 312 and 389. To understand the role of p53 phosphorylation, we generated p53 constructs encoding serine-to-alanine or serine-to-glutamate mutations at these codons. The transcriptional activity were measured in cells capable of contact inhibition. In low-density cycling cells, no difference in transcriptional activity was found between wild type p53 and any of the mutants. In contact-inhibited cells, however, only mutations of p53 at serine 389 resulted in altered responses to cell cycle arrest and to cyclin E overexpression. The mutant with serine-to-glutamate substitution at codon 389 retained its function in contact inhibited cells. Cyclin E overexpression in these cells induced p53 phosphorylation at serine 389. Furthermore, we showed that phosphorylation at serine 389 regulates p53 DNA binding activity. Our findings implicate that phosphorylation is an important mechanism for p53 activation.^ p53 is the most frequently mutated gene in human tumors. To study the mechanism of p53 inactivation by mutations, we carried out detailed analysis of a murine p53 mutation with an arginine-to-tryptophane substitution at codon 245. The corresponding human p53 mutation at amino acid 248 is the most frequently mutated codon in tumors. We showed that this mutant is inactive in suppressing focus formation, binding to DNA and transactivation. Structural analysis revealed that this mutant assumes the wild type protein conformation. These findings define a novel class of p53 mutations and help to understand structure-function relationship of p53. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

{\it cis\/} -acting determinants in the control of MuSVts110 RNA splicing

Like other simple retroviruses the murine sarcoma virus ts110 (MuSVts110) displays an inefficient mode of genome splicing. But, unlike the splicing phenotypic of other retroviruses, the splicing event effected upon the transcript of MuSVts110 is temperature sensitive. Previous work in this laboratory has established that the conditionally defective nature of MuSVts110 RNA splicing is mediated in cis by features in the viral transcript. Here we show that the 5$\sp\prime$ splice site of the MuSVts110 transcript acts as a point of control of the overall splicing efficiency at both permissive and nonpermissive temperatures for splicing. We strengthened and simultaneously weakened the nucleotide structure of the 5$\sp\prime$ splice site in an attempt to elucidate the differential effects each of the two known critical splicing components which interact with the 5$\sp\prime$ splice site have on the overall efficiency of intron excision. We found that a transversion of the sixth nucleotide, resulting in the formation of a near-consensus 5$\sp\prime$ splice site, dramatically increased the overall efficiency of MuSVts110 RNA splicing and abrogated the thermosensitive nature of this splicing event. Various secondary mutations within this original transversion mutant, designed to selectively decrease specific splicing component interactions, lead to recovery of inefficient and thermosensitive splicing. We have further shown that a sequence of 415 nucleotides lying in the downstream exon of the viral RNA and hypothesized to act as an element in the temperature-dependent inhibition of splicing displays a functional redundancy throughout its length; loss and/or replacement of any one sequence of 100 nucleotides within this sequence does not, with one exception detailed below, diminish the degree to which MuSVts110 RNA is inhibited to splice at the restrictive temperature. One specific deletion, though, fortuitously juxtaposed and activated cryptic consensus splicing signals for the excision of a cryptic intron within the downstream exon and markedly potentiated--across a newly defined cryptic exon--the splicing event effected upon the upstream, native intron. We have exploited this mutant of MuSVts110 to further an understanding of the process of exon definition and intron definition and show that the polypyrimidine tract and consensus 3$\sp\prime$ splice site, as well as the 5$\sp\prime$ splice site, within the intron at the 3$\sp\prime$ flank of the defined exon are required for the exon's definition; implying that definition of the downstream intron is required for the in vivo definition of the proximal, upstream exon. Finally; we have shown, through the construction of heterologous mutants of MuSVts110 employing a foreign 3$\sp\prime$ end-forming sequence, that efficiency of transcript splicing can be increased--to a degree which abrogates its thermosensitive nature--in direct proportion to increasing proximity of the 3$\sp\prime$ end-forming signal to the terminal 3$\sp\prime$ splice site. ^

Creation of a retinoblastoma mutant with dominant -negative activity

The Retinoblastoma tumor suppressor gene (RB) plays a role in a variety of human cancers. Experimental analyses have indicated that the protein product of the RB gene (pRb) plays a role in cell cycle regulation, and that this protein is required in cellular differentiation, senescence, and cell survival. pRb function is dependent on its ability to bind to cellular factors. There are multiple protein binding domains within pRb. Mutations within these domains which eliminate the ability of pRb to bind its targets result in loss of function. Loss of pRb function leads to tumorigenesis, although uncontrolled cellular proliferation is not a universal response to pRb inactivation. The ultimate response to the loss of pRb is influenced by both the genetic and epigenetic environments. Targeted disruption of RB in mice results in embryonic lethality, demonstrating the requirement for functional pRb in development. Close examination of various tissues from the embryos which lack wildtype RB shows problems in differentiation as well as showing induction of apoptosis. Although disruption of RB has provided useful information, complete inactivation of a gene precludes the possibility of discovering the functions that separate domains may have within the system. Creation of a dominant negative mutant by domain deletion whose phenotype is expressed in the presence of the wildtype may provide information about the intermediate functions of the protein. In addition, tissue specific targeting of a dominant negative mutant of pRb allows for comprehensive analysis of pRb function in organogenesis. In this thesis, a series of RB deletion mutants were created and tested for dominant negative activity as well as cellular localization. A tissue culture assay for dominant negative activity was developed which screens for the phenotype of apoptosis due to loss of pRb function. Two mutants from this series scored positive for dominant negative activity in this assay. The effect of these mutants within the assay environment can be explained by a model in which pRb acts as a facilitator of cell fate pathway decisions. ^

Signal transduction of {\it HER-2\/}/{\it neu\/} receptor tyrosine kinase

Overexpression and/or amplification of HER2/neu is frequently detected in many human cancers. Activation of p185 tyrosine kinase can be achieved by point mutation, overexpression, deletion, and heterodimerization with other class I receptors. In this study I investigated the signal transduction pathways mediating the oncogenic signal of the point mutation-activated rat p185. I demonstrated that tyrosine phosphorylation of Shc and formation of Shc/Grb2 complex correlated to the transformation of NIH3T3 cells caused by the point mutation-activated rat HER2/neu. Furthermore, I observed that association with Shc was severely impaired by deletion of most of the major autophosphorylation sites of the point-mutated p185. The truncated p185 product, however, fully retained its ability to transform NIH3T3 cells, induce Shc tyrosine phosphorylation and Shc/Grb2 complex formation. These results suggest that tyrosine phosphorylation of Shc which allows formation of Shc/Grb2 complex may play an important role in cell transformation induced by the point mutation-activated p185, and that stable binding to mutant p185 may not be necessary for Shc to mediate this signaling pathway.^ Recent studies have suggested that formation of the complex containing Sos, Grb2 and Shc is important in coupling receptor tyrosine kinases to the Ras signaling pathway. To clarify the role of this trimer in the oncogenic signaling of the activated p185, I set out to interfere with the protein-protein interactions in Shc/Grb2/Sos complex by introducing Grb2 mutants with deletions in either amino- ($\Delta$N-Grb2) or carboxyl- ($\Delta$C-Grb2) terminal SH3 domains into B104-1-1 cells derived from NIH3T3 cells that express the point mutation-activated HER-2/neu. I found that the transformed phenotypes of the B104-1-1 cells were largely reversed by expression of the $\Delta$N-Grb2. The effect of the $\Delta$C-Grb2 on phenotypic reversion was much weaker. Biochemical analysis showed that the $\Delta$N-Grb2 was able to associate Shc but not the activated p185 nor Sos, while the $\Delta$C-Grb2 bound to Shc, the activated p185, and Sos. The p185-mediated Ras activation was severely inhibited by the $\Delta$N-Grb2 but not the $\Delta$C-Grb2. Taken together, these data demonstrate that interruption of the interaction between Shc and the endogenous Grb2 by the $\Delta$N-Grb2 is able to impair the oncogenic signaling of the mutation-activated p185, indicating that (i) the $\Delta$N-Grb2 functions as a strong dominant-negative mutant, (ii) Shc/Grb2/Sos pathway plays a major role in mediating the oncogenic signal of the mutation-activated p185. Unlike the $\Delta$N-Grb2, the $\Delta$C-Grb2 appears to be a relatively weak dominant-negative mutant, probably due to its ability to largely fulfill the biological functions of the wild-type Grb2. ^

Regulation of apoptosis during the pathogenesis of non -melanoma skin cancer

Previous studies from our lab have shown distinctive patterns of expression of bcl-2 gene family members in human nonmelanoma skin cancer (NMSC). To further evaluate the significance of these observations and to study the effects of cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic protein expression was confirmed in all the layers of the epidermis except the stratum corneum using immunohistochemistry. Multifocal epidermal hyperplasia, without associated hyperkeratosis, was observed in newborn HK1.bcl-2 mice. Immunofluorescence staining using monoclonal antibodies specific for a variety of differentiation markers revealed aberrant expression of keratin 6 (K6) in the transgenic epidermis. Epidermal proliferative indexes, assessed by anti-BrdUrd immunofluorescence staining, were similar in control and transgenic newborn mice, but suprabasal proliferating cells were seen within the hyperplastic areas of the transgenic mouse skin. Spontaneous apoptotic indices of the epidermis were similar in both control and HK1.bcl-2 transgenic newborn mice, however, after UV-B irradiation, the number of "sunburn cells" was significantly higher in the control compared to the HK1.bcl-2 transgenic animals.^ Adult HK1.bcl-2 and control littermate mice were used in UV-B and chemical carcinogenesis protocols including DMBA + TPA. UV-B irradiated control and HK1.bcl-2 mice had comparable incidence of tumors than the controls, but the mean latency period was significantly shorter in the HK1.bcl-2 transgenic. Both control and transgenic animals included in chemical carcinogenesis protocols required application of both the initiating (DMBA) and promoting (TPA) agents to develop tumors. The frequency, number, and latency of tumor formation was similar in both groups of animals, however, HK1.bcl-2 mice exhibited a rate of conversion from benign papilloma to carcinoma 2.5 times greater than controls.^ Similar carcinogenesis experiments were performed using newborn mice. HK1.bcl-2 mice treated with UV-B plus TPA have a three fold greater incidence of tumor formation compared to controls littermates. HK1.bcl-2 transgenic animals also exhibited a shorter latency for papilloma formation when treated with DMBA plus TPA.^ HK1.bcl-2/v-Ha-ras double transgenic mice shared phenotypic features of both HK1.v-Ha-ras and HK1.bcl-2 transgenic mice, and exhibited focal areas of augmented hyperplasia. These double transgenic mice were susceptible to tumor formation by treatment with TPA alone.^ Cultures of primary keratinocytes were established from control, HK1.bcl-2, HK1.Ha-ras, and HK1.bcl-2/v-Ha-ras newborn mice. Cell viability was determined after exposure of the cells to UV-B irradiation, DMBA, TPA, or TGF-$\beta$1. Internucleosomal DNA fragmentation ("ladders") and morphological cellular changes compatible with apoptotic cell death were observed after the application of all these agents. HK1.bcl-2 keratinocytes were resistant to cell death induction by all of these agents except TGF-$\beta$1. HK1.Ha-ras cells had a higher spontaneous rate of cell death which could be compensated by co-expression of bcl-2.^ These findings suggest that bcl-2 dependent cell death suppression may be an important component of multistep skin carcinogenesis. ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

The identification and regulation of a novel interaction occurring between $\beta$-catenin and the actin -bundling protein fascin

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesion, catenins have more recently been indicated to participate in cell and developmental signaling pathways. $\beta$-catenin, for example, associates directly with receptor tyrosine kinases and transcription factors such as LEF-1/TCF, and tranduces developmental signals within the Wnt pathway. $\beta$-catenin also appear to a role in regulating cell proliferation via its interaction with the tumor supressor protein APC. I have employed the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to $\beta$-catenin's central Armadillo-repeat domain. The $\beta$-catenin-fascin interaction exists in cell lines as well as in animal brain tissues as revealed by immunoprecipitation analysis, and substantiated in vitro with purified proteins. Fascin additionally binds to plakoglobin, which contains a more divergent Armadillo-repeat domain. Fascin and E-cadherin utilize a similar binding-site within $\beta$-catenin, such that they form mutually exclusive complexes with $\beta$-catenin. Fascin and $\beta$-catenin co-localize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. Total immunoprecipitable b-catein has several isoforms, only the hyperphosphorylated isoform 1 associated with fascin. An increased $\beta$-catenin-fascin interaction was observed in HGF stimulated cells, and in Xenopus embryos injected with src kinase RNAs. The increased $\beta$-catenin association with fascin is correlated with increased levels of $\beta$-catenin phosphorylation. $\beta$-catenin, but not fascin, can be readily phosphorylated on tyrosine in vivo following src injection of embryos, or in vitro following v-src addition to purified protein components. These observations suggest a role of $\beta$-catenin phosphorylation in regulating its interaction with fascin, and src kinase may be an important regulator of the $\beta$-catenin-fascin association in vivo. The $\beta$-catenin-fascin interaction represents a novel catenin complex, that may conceivably regulate actin cytoskeletal structures, cell adhesion, and cellular motility, perhaps in a coordinate manner with its functions in cadherin and APC complexes. ^