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Breast cancer is a highly diverse group of cancers and consists of at least 5 different subgroups. Furthermore, patients with Intra-tumor heterogeneity due to the presence of cancer cells with variable phenotypes such as different degrees of basal-like and luminal features increase the complexity of treatment. The aim of this project is to better understand the mechanisms that regulate breast cancer cell plasticity and origins of breast cancer heterogeneity. Our focus is the function of protein kinase D1 (PKD1), which is a serine/threonine kinase. We previously showed that PKD1 can repress epithelial to mesenchymal transition (EMT) by inhibitory phosphorylation of transcription factor Snail, a master switch of EMT. Supported by this award, we have performed experiments on molecular, cellular, mice xenografts and transgenic levels and conclude that PKD1 is a context-dependent tumorigenesis and metastasis repressor or enhance. Specifically, PKD1 is a metastasis repressor in luminal type breast cancer and loss of PKD1 in luminal type cells converts them into basal-like cells; and PKD1 is an enhancer of tumorigenesis in basal-like breast cancer. Based on current available data, conditional knockout PKD1 in mouse mammary tissue does not disrupt mammary development and does not induce tumorigenesis., The original document contains color images.

The focus of this proposal is Neuropilin-2 (NRP2), a VEGF receptor that is not expressed in normal prostate but is expressed in prostate cancer and correlates with Gleason grade. We demonstrated that PTEN deletion induces NRP2 expression and propose that NRP2 contributes to the function of prostate cancer stem cells and tumor formation. We also discovered that NRP2 facilitates the expression of Bmi-1, a transcriptional repressor, and that NRP2 suppresses the IGF-1 receptor (IGF-1R) by a mechanism that involves transcriptional repression by Bmi-1. We have obtained preliminary evidence that targeting NRP2 directly on tumor cells in combination with IGF-1R inhibition is effective treating aggressive prostate carcinoma and pursuing this possibility more rigorously. The role of VEGF/NRP2 signaling in the function of prostate cancer stem cells and tumor initiation is also being investigated., The original document contains color images.

Breast cancer develops from epithelial lesions in breast ducts and lobules that become invasive and can be metastatic. Breast cancer is a disease of uncontrolled cell division. Cell division normally creates two genetically identical daughter cells through severing of a cytoplasmic bridge that interconnects them. The midbody is an organelle involved in severing. Previously midbodies were thought to be lost from cells after division, but we show they can be retained. Here we show that MBs exhibit stem cell properties and are in breast cancer stem cells (Task 1). They are scaffolds for anchoring breast cancer oncogenes, tumor suppressors and breast cancer stem cell proteins. Increasing MB+ cells increases in vitro tumor potential ( Task 2). We activated a MB-degradation pathway that decreased MBs and tumorigenic properties of breast cancer cells (Task 3). Because MBs are in breast cancer stem cells, we believe this MB targeting strategy may be an innovative strategy for therapies focused on the most difficult of all tumors, those thought to be caused by cancer stem cells, namely recurrent, resistant and metastatic cancers., The original document contains color images.

Breast cancer is a complex disease that develops from epithelial lesions confined to breast ducts and lobules and progresses rapidly to become locally invasive and finally metastatic. Our recent studies show that breast cancer cells undergo asymmetric events during cell division that generate different daughter cells. One daughter receives the singular midbody (MB) that is made during every cell division. The cell with this so-called postmitotic midbody derivative accumulates additional MBds with successive divisions. In breast tumor sections, rare cells stain for MBds adjacent to the basal layer, the position of putative breast cancer stem cells. MBds are present in high numbers in several human breast cancer cell lines and in human tumors, but are rarely found in normal breast epithelial cell lines or breast tissue. MBds are also found in several well-characterized mouse and human stem cell niches but not in adjacent transit amplifying or differentiating cells. These results suggest that MBds are in almost exclusively in stem cells and putative breast cancer stem cells (CSS). This idea is consistent with the emerging view that breast cancer develops from transformation of stem cells. Based on these observations, we propose that MBds 1) will serve as markers for breast CSCs, 2) may have diagnostic/prognositc value for breast cancer progression and 3) could directly contribute to breast carcinoma. To test this, we propose the following aims: 1) Quantify MBds in breast tumors and cell lines and compare with normal breast epithelial cells. 2) Test MBd-containing breast cancer cells for CSC activities in vivo and in vitro. 3) Test MBds for their ability to confer breast cancer stem cell properties by disrupting MBd inheritance or RNAi.

Breast cancer is a complex disease that develops from epithelial lesions confined to breast ducts and lobules and progresses rapidly to become locally invasive and finally metastatic. Our recent studies show that breast cancer cells undergo asymmetric events during cell division that generate different daughter cells. One daughter receives the singular midbody (MB) that is made during every cell division. The cell with this so-called postmitotic midbody derivative accumulates additional MBds with successive divisions. In breast tumor sections, rare cells stain for MBds adjacent to the basal layer, the position of putative breast cancer stem cells. MBds are present in high numbers in several human breast cancer cell lines and in human tumors, but are rarely found in normal breast epithelial cell lines or breast tissue. MBds are also found in several well-characterized mouse and human stem cell niches but not in adjacent transit amplifying or differentiating cells. These results suggest that MBds are in almost exclusively in stem cells and putative breast cancer stem cells (CSS). This idea is consistent with the emerging view that breast cancer develops from transformation of stem cells. Based on these observations, we propose that MBds 1) will serve as markers for breast CSCs, 2) may have diagnostic/prognositc value for breast cancer progression and 3) could directly contribute to breast carcinoma. To test this, we propose the following aims: 1) Quantify MBds in breast tumors and cell lines and compare with normal breast epithelial cells. 2) Test MBd-containing breast cancer cells for CSC activities in vivo and in vitro. 3) Test MBds for their ability to confer breast cancer stem cell properties by disrupting MBd inheritance or RNAi., The original document contains color images.

Breast cancer is a complex disease that develops from epithelial lesions confined to breast ducts and lobules and progresses rapidly to become locally invasive and finally metastatic. Our recent studies show that breast cancer cells undergo asymmetric events during cell division that generate different daughter cells. One daughter receives the singular midbody (MB) that is made during every cell division. The cell with this so-called postmitotic midbody derivative accumulates additional MBds with successive divisions. In breast tumor sections, rare cells stain for MBds adjacent to the basal layer, the position of putative breast cancer stem cells. MBds are present in high numbers in several human breast cancer cell lines and in human tumors, but are rarely found in normal breast epithelial cell lines or breast tissue. MBds are also found in several well-characterized mouse and human stem cell niches but not in adjacent transit amplifying or differentiating cells. These results suggest that MBds are in almost exclusively in stem cells and putative breast cancer stem cells (CSS). This idea is consistent with the emerging view that breast cancer develops from transformation of stem cells. Based on these observations, we propose that MBds 1) will serve as markers for breast CSCs, 2) may have diagnostic/prognositc value for breast cancer progression and 3) could directly contribute to breast carcinoma. To test this, we propose the following aims: 1) Quantify MBds in breast tumors and cell lines and compare with normal breast epithelial cells. 2) Test MBd-containing breast cancer cells for CSC activities in vivo and in vitro. 3) Test MBds for their ability to confer breast cancer stem cell properties by disrupting MBd inheritance or RNAi., The original document contains color images.

The purpose is to define the relationship between neurotensin (NT) and protein kinases and to investigate the mechanism by which flavonoids (FLAV) inhibit NT growth signaling in PC3 cells. The long-range scope is to determine the significance of NT in the negative effects of high fat intake on PC incidence and the positive effects of diets containing FLAV. Our results show that NT-induced growth signaling involves activation of PKC, that arachidonic acid metabolism and EGF receptor activation participate in the NT signaling process, that cell metabolism and ATP levels can influence NT receptor function, and that activated PKC (most notably PKC alpha and beta) can feed back to regulate the ability of NT receptor to bind NT and to initiate signaling. FLAV was found to exert differential effects on PKC isotype activation and downregulation. In addition, FLAV was shown to inhibit protein tyrosine kinase activity in PC3 cells and evidence supports the notion that this can input to regulate NT receptor function. NT receptor was shown to localize in caveolae and to be displaced when activated by NT. FLAV appeared to influence this response to NT. These findings have implications regarding mechanisms that regulate NT receptor function and the design of agents to block NT-induced growth signaling in PC., The original document contains color images.

We showed that zinc treatment induced MT expression in LNCaP and C4-2 PCa cells as determined by Western blotting and DNA microarray analysis. Chemotherapy and radiation sensitivity assays of cells after treatment with cisplatin or radiation were performed in the presence, or absence, of 150 microM ZnSO4, and cell viability was measured after 72 hours by MTS viability and clonogenic and flow cytometry assays. The experiments were repeated three times and the data analyzed. We found that increasing concentrations of ZnSO4 upregulated MT expression in a dose-dependent manner. Microarray analysis demonstrated a specific increase in MT expression. Cells treated with zinc demonstrated a significantly decreased sensitivity to cisplatin and radiotherapy compared with controls (P <0.05). Our data have confirmed that treatment of PCa with zinc causes an increase in MT expression, which is significantly associated with resistance to cisplatin chemotherapy and radiotherapy in prostate cancer. Therapeutic targeting of MT may therefore provide a means to overcome resistance to radiotherapy and cisplatin chemotherapy in prostate cancer., The original document contains color images.

The purpose is to define the relationship between neurotensin (NT) and protein kinase C (PKC) isotypes and to investigate the mechanism by which flavonoids (FLAV) inhibit NT growth signaling in PC3 cells. The long-range scope is to determine the significance of NT in the negative effects of high fat intake on PC incidence and the positive effects of diets containing FLAV. Our results show that NT-induced growth signaling involves and requires activation of several PKC isotypes (most notably PKC epsilon and delta) that arachidonic acid metabolism and EGF receptor activation participate in the NT signaling process that cell metabolism and ATP levels can influence NT receptor function and that activated PKC (most notably PKC alpha and beta) can feed back to regulate the ability of NT receptor to bind NT and to initiate signaling. FLAV was found to exert differential effects on PKC isotype activation and downregulation. Thus FLAV could alter the balance between conventional and novel PKC acbvity which could influence growth responses to NT. These findings have implications regarding mechanisms that regulate NT receptor function and the design of agents to block NT-induced growth signaling in PC., The original document contains color images.

The VEGF receptor Flt-1 mediates VEGF survival signaling in cancer cells and has been correlated with a high risk metastasis and relapse in breast cancer. However, the function of Flt-1 in breast cancer is not yet known. Here we report an increase in Flt-1 expression in breast tumor cells exposed to a hypoxic environment. Interestingly, no increase in Flt-1 expression was observed in pre-malignant breast epithelial cells. Furthermore, Flt-1 was identified as a target of miR-10b, which is deregulated in breast cancer. We find that expression of mature miR-10b in breast cancer cells results in significant down-regulation of Flt-1 expression. These findings will serve as a basis for functional studies of the role of Flt-1 and miR-10b in the etiology of breast cancer., The original document contains color images.

The goal of this investigation is to using anti-Her2 antibody Herceptin as carrier and streptavidin as linker to specifically transport antisense into tumor cells expressing Her2. For this purpose we successfully conjugated MAG3/biotin to antisense/sense morpholino oligomers (MORFs) and conjugated biotin group to Herceptin. The MORF-streptavid in-Herceptin constructs have been synthesized and their quality has been confirmed. We have confirmed by confocal microscopy using fluorescent lissamine as the tag that Herceptin can mediate the cellular internalization of MORFs and more important the internalized oligomer distributed in cytoplasm evenly without apparent entrapment in cellular compartments. By using 99mTc as the tag we also have approved that the antisense can be released from the internalized antisense-streptavidin-Herceptin construct. These results are significant and encouraging for the followed studies in in vivo antisense tumor targeting using Herceptin as carrier., The original document contains color images.

The purpose is to define the relationships between neurotensin (NT) and protein kinase C (PKC) isotypes and to investigate the mechanism by which flavonoids (FLAV) inhibit NT-induced signaling in PC3 cells. The long-range scope is to determine the significance of NT as a participant in the negative effects of high fat intake on PC incidence and growth, and the positive effects of diets containing large amounts of FLAV. Our results show that NTinduced growth signaling in PC3 cells involves and requires the activation of several PKC isotypes, that arachidonic acid release and lipoxygenase activity participate in the signaling process, that cellular metabolism and ATP levels are important inputs to NT receptor function, and that activated PKC feeds back to regulate the ability of NT receptor to bind and to initiate signaling. These findings have implications regarding general mechanisms of G protein-linked receptor function and the design of new agents to block NT-induced growth signaling in PC., The original document contains color images.

Breast cancer is the most prevalent of all cancers and it is the second most common cause of cancer death amongst women. Centrosome defects have been implicated in cancer formation and can be detected very early in this process. The centrosome protein pericentrin is overexpressed in many cancers including breast cancer. We found that down regulation of pericentrin leads to cytokinesis defects and aneuploidy. Pericentrin overexpression leads to an increase in the number of multinucleated cells, suggesting a defect in cytokinesis. In addition to the cytokinesis defects, pericentrin knock down also induce a G0/G1 arrest that is p53-p38 dependent. Moreover, pericentrin knockdown affects cilia formation in epithelial cells. Also, knock down of several other centrosome proteins leads to aG1 arrest and affects cilia formation in epithelial cells. We conclude that pericentrin affects cytokinesis, G1progression and differentiation. Defects during mitosis, as well as loss of checkpoints and uncontrolled cell division are the first steps in cancer formation. Understanding those events will be a major advance in breast cancer research.

Aims: (a) study mechanism of neurotensin(NT)-enhanced prostate cancer(PC)growth; (b)test if NT released from gut contributes to the PC-growth effect of fatty diets. In PC3 cells, NT transactivated EGF receptor (EGFR), liberating HB-EGF from cell-surface by a protease-dependent and PKC-dependent mechanism. Activation of EGFR led to activation of MAP-kinase, P13-kinase, AKT and p7O-S6-kinase, and in 24 hrs to enhanced DNA synthesis. NT and EGF stimulated cellular release 3H-AA. Inhibitors of Lipoxygenase (LOX) blocked basal and NT- induced DNA synthesis, suggesting that EGF and NT acted by way of PLA2, S-LOX and 12-LOX (present by western blot). NT receptor function was modulated by antioxidants and calcium channel blockers, which indirectly blocked inositol phosphate formation. In the presence of a Gs stimulus, NT enhanced cAMP production, giving an inhibition of DNA synthesis. Thus, NT effects involve EGFR, protein kinases, LOXs and adenylyl cyclases, and its effects depend on the hormonal milieu. An NT-knockout-nude mouse strain was developed and PC3 cell xenografts were found to grow faster in NT(++) mice than in NT(--) mice given a high fat diet. Our results suggest that NT released during fat ingestion stimulates PC growth via effects involving EGFR, protein kinases, LOXs and calcium channels.

Chromatin structure has a strong influence on the regulation of all nuclear processes in which access to DNA is required, such as transcription, replication and DNA repair. We have previously shown that Rad54p, a member of the SWl2/SNF2 family required for the repair of DNA double-strand breaks (DSBs) by homology recombination, is a bona fide ATP-dependent chromatin remodeling enzyme, and that the ability of Rad54 to modulate chromatin structure is required for the proper repair of DSBs in a chromatin context. We had previously observed that Rad54p was stabilized against thermal denaturation by chromatin. Here, we report that this stabilizating effect is real and specific, and it can be attributed to the ability of Rad54p to directly interact with the amino-terminal portion of hi stones H3 and H4. Interestingly, H3 and H4 amino-terminal domains are targets of post-translational modifications required for DNA repair. Particularly, H3 acetylation by Hatlp, and H4 acetylation by Esalp have been shown to be critical steps in the repair of DSBs. We found that even though Rad54p discriminately binds H3 and H4 proximal amino-terminal domains, this interactions are not essential for its ability to remodel chromatin and drive the homologous recombination process in our in vitro setup.

The second annual report of progress in the study entitled A holistic quality of life intervention for patients with secondary prostate cancer' is contained. Each of the tasks listed in the approved statement of work' that were applicable to the second year of the study is contained. In particular: Task 1: Patient identification and referral. Task 2: Patient contact, intake of patients into study, patient tracking. Task 3 Review of intervention group meetings and materials. The successful completion of two intervention cycles and the subsequent three-month follow-up data collection procedures is described. Special attention is given to the adapted recruitment strategies that have been developed to remedy the shortfall of study accrual numbers for recruitment into the third intervention cycle that begins in March 2004. Also included are the completed participant intervention manual and accompanying CD. The featuring of this study on the ABC national evening network news broadcast as an example of an innovative approach to cancer treatment is also discussed. Because the intervention period of this study is not yet complete no outcomes are reported., The original document contains color images. All DTIC reproductions will be in black and white.

In this study, neuroprotective ganglioside derivatives are examined so they can be targeted to specific points in cell death pathways. Here, ganglioside functional groups required for neuroprotection and blood-brain barrier permeance are determined. Cell death mechanisms and the mechanism(s) by which semisynthetic gangliosides intervene in the cell death process are also studied. In the second year C2, C4, CS, C14, C20, and C26 fatty acid GMl derivatives were synthesized. Experiments on semisynthetic ganglioside cytoprotection using a retinoic acid- differentiated SH-SY5Y human neuroblastoma cell/MPP+ model have been initiated. Each derivative requires analysis at different concentrations and preincubation times. Of the compounds studied to date (C2GM1, dichloroC2GM1, C4GM1, Cl4GM1), the C4GM1 derivative is the most cytoprotective, being 1.4 times more effective than the+ parent GM1. cDNA microarray analysis of changes in gene expression associated with MPP toxicity revealed that MPP+ exposure resulted in decreased RMB3 expression, increased GADD153 expression, and increased c-Myc expression. Further findings suggest that increased pancreatic protein disulfide isomerase (PDIp) expression in MPP+ stressed SH-SY5Y cells may promote Lewy body formation and perhaps contribute to neurotoxicity and neurodegeneration., The original document contains color images. All DTIC reproductions will be in black and white.

Despite the historical use of estrogen in the treatment of PCa little is known concerning the direct biological effects they have on the prostate, what role they may play in carcinogenesis of the gland and what mechanisms mediate the therapeutic effects of these hormones/antihormones on PCa. Therapies for advanced prostate treatment are limited and effective chemopreventive strategies for PCa are lacking. The consortium projects collectively offer a comprehensive and unique approach to address these needs. Projects are selected based on their high relevance to PCa treatment and prevention, creativities in applying the state- of-the-art technologies, experience and the willingness of the investigator to work in a synergistic manner, and short translational time-frame for bringing the end products to the clinics. Three consortium projects are focused on mechanism by which estrogens may play a role in prostatic carcinogenesis. These include studies seeking to 1) identifying genetic polymorphisms of CYP1B1 that contribute to PCa risk and race-based disparity in incidence, 2) epigenetic mechanisms for silencing ER-beta, during prostate carcinogenesis, and 3) estrogen-mediated oxidative DNA damage and its prevention by antioxidants. Results from these studies may yield new diagnostic genetic tests for PCa, identify high-risk individuals and populations, and for formulating novel preventative/intervention strategies. The other three projects are directly aimed at the treatment of advanced PCa. One is concerned with the synthesis, efficacy testing and defining mechanisms of action of a new class of estrogen-based therapeutic agents. Another is a comprehensive genetic and biological investigation of the herbal-based phytoestrogen formulation, PC-SPES, commonly used as alternative medicine by PCa patients, and the last is a clinical Phase II trial to test the efficacy of DES and Faslodex in the treatment of androgen-independent PCa.

Chromosomal double-strand breaks (DSBs) can arise from cellular DNA metabolism and from eogenous DNA-damaging agents. Efficient repair of these DSBs is critical for genome stability. Rad54, a member of the SWI2/SNF2 family, is involved in the homology recombination (ER) DNA repair pathway. Rad54 has been shown to physically associate with Rad51, a eukaryotic recombinase that in mammals interacts with tumor suppressors BRCA1 and BRCA2.

Centrosomes are essential organelles that organize mitotic spindles for chromosome segregation and assemble interphase microtubules for controlling cell shape/polarity. All these functions are disrupted in carcinomas, implicating centrosomes in tumorigenesis. We and another group were first to discover that centrosomes were abnormal in most malignant human carcinomas. The work accomplished in this proposal provides evidence for a causative role for centrosomes in prostate cancer., The original document contains color images. All DTIC reproductions will be in black and white.

In this study, neuroprotective ganglioside derivatives are examined so they can be targeted to specific points in cell death pathways. GM1 ganglioside and several of its chemically modified derivatives are neuroprotective in several neurotoxic models. Here, ganglioside functional groups required for neuroprotection and blood-brain barrier (BBB) permeance are determined Cell death mechanisms are also defined, as are the mechanism(s) by which ganglioside derivatives intervene in the cell death process. In the first year, substantial quantities of GM1 have been isolated and purified., The original document contains color images. All DTIC reproductions will be in black and white.

Our goal is to understand mechanisms by which neurotoxicity destroys cells in the substantia nigra. Our hypothesis is that c-JUN kinases (JNK) which can contribute to neuronal death mediate neurodegeneration in the substantia nigra after exposure to MPTP or excitotoxicity. Results in year 3 indicated that mice lacking JNK 1 or JNK 3 lack neuroprotection against MPTP neurotoxicity in the substantia nigra and excitotoxicity in striatal neuronal cultures. Results support a role for JNK proximal to mitochondrial initiation of apoptosis and overlapping function of JNK isoforms. Furthermore, in another model of neuronal apoptosis p38 and not JNK serves as a target for MAP kinase dependent apoptosis. Additional studies indicate that neuronal apoptotic mechanisms can lead to increased aggregation of proteins in selected neurodegenerative diseases. Preliminary studies on stem cell transplantation were extended to show that knockout of an immune related gene improves survival and integration of stem cells in substantia nigra. Our studies offer a revised signaling system for neuronal apoptosis and new approach to improve survival of bone-marrow derived stem cells in damaged brain.

The aims are (a) to determine the mechanism by which neurotensin (NT) enhances prostate Cancer (PC3) cell growth and (b) to test if NT released by eating mediates the cancer- Promoting effects of high fat diets Experiments conducted here show that NT enhanced Arachidonic acid metabolism generating primarily lipoxygenase-derived products NT Stimulated DNA-replication and its effect was blocked by lipoxygenase inhibitors by MAP-kinase inhibitors by PI3 kinase inhibitor, by Ca2+ - channel blocker and by tyrosine Kinase inhibitors. NT caused the accumulation of EGF-like ligand(s) and transactivation Of the EGF receptor.

The goal of this project was to identify peptides from phage display peptide libraries which bind with high affinity to the mutant EGFRvIII receptor present in breast tumors. The peptides selected were radiolabeled with technetium-99m (99mTc) and tested for their potential as agents in the detection of breast cancer. Using available phage display peptide libraries, we have identified five consensus peptides that show affinity for cells expressing the mutant EGFRvIII receptor. Characterization of these selected peptides was by ELISA and radiolabeled cell binding studies. First, the labeled phage were tested in in vitro assays and in mice with tumors. Specific binding of The labeled phage to the study cells was found relative to the control cells. Also, mice with tumors expressing the mutant receptor showed enhanced accumulation of the labeled phage over mice with tumors expressing the wild-type receptor. The consensus peptides were identified through analysis of the phage DNA. The peptides were synthesized, then conjugated to a chelator for radiolabeling with 99mTc. All peptides have been tested in in vitro assays and tested in tumor bearing mice. The in vivo studies show that the 99mTc peptide clear the circulation quickly and demonstrate accumulation in breast tumor. Peptides have also been evaluated against a panel of tumor from clinical pathology. Early results suggest a distinction of peptides for various tumors.

To investigate the role of ER-beta in human prostate carcinogenesis, and in growth regulation of a prostate cancer cell line (DU145), we proposed: 1) to study effects of antiestrogens/estrogens on growth of DU145 cells, 2) to Immunostain tissue sections from discarded human radical prostatectomy specimens and to perform semiquantitative RT-PCR on microdissected tissues to determine the level of ER-beta expression in normal and abnormal prostate tissues. Under Specific Aim 1, the proposed works have been completed and the results had been published in Cancer Research Journal and reported in the first progress report. In addition, we demonstrated the antiestrogen-induced cell growth inhibition in DU145 cells might be due to the induction of cell cycle arrest at GO-GI phase. Under Specific Aim 2, Using G017 we showed that in normal prostates, ER-beta was strongly expressed in the nuclei of basal epithelial cells. ER-beta expression was transiently elevated in early PIN lesions, but progressively diminished in higher grade PIN lesions. Similarly, ER-beta immunostaining was markedly diminished in Gleason grade 3 and 4 adenocarcinomas and absent in higher-grade cancers. ER-beta expression reappeared in metastatic prostatic carcinomas. The results were also compared to ER-alpha and androgen receptor expressions in those samples.

Centrosomes are essential organelles that control mitotic spindle organization, chromosome segregation, cell shape and cell polarity--all features of epithelial gland integrity. In the studies supported by this grant, we (and another group) discovered that centrosomes were structurally and numerically abnormal in nearly all malignant human tumors. Moreover, artificial induction of centrosome defects--by elevating the levels of a single centrosome protein called pericentrin--caused cellular disorganization and genomic instability in nontumor cells. Examination of prostate tumor tissues showed that pericentrin levels were elevated and the levels increased during tumor progression. We consistently found that cells with elevated pericentrin levels had centrosome defects and genomic instability. Finally, centrosome defects were detected in a percentage of precursor lesions of prostate carcinoma (approximately equal 20% of PIN lesions) together with genetic instability. Taken together, these results suggest that centrosome defects and elevated pericentrin levels contribute to rather than result from the tumorigenic process. Centrosome defects may thus prove to be a prognostic indicator of aggressive disease., Original contains color plates: All DTIC reproductions will be in black and white.

The goal of this project is to select for a peptide, using phage display libraries, which recognizes an EGF mutant receptor present in tumor cells. Phage display is a methodology which offers great potential for cancer diagnostic agents. The peptide(s) selected will be radiolabeled with technetium-99m and tested in vitro and in in vivo mouse tumor models for their potential as agents for detection, through nuclear imaging, of breast cancers which expresses the mutant EGF receptor. If successful, this Tc4abeled mutant EGF-binding protein could serve not only as a useful agent in the diagnosis of breast cancer through imaging, but potentially be applicable to other cancers as well. Although not part of this proposal, the novel peptide selected as may be useful in therapy, by serving as a targeting agent in the delivery of therapeutic cancer drugs directly to cancer cells.

Women with breast cancer who receive psychosocial interventions may have longer disease-free and total survival. Psychological distress seems to be particularly acute in younger women with breast cancer, a population that seems particularly amenable to psychosocial interventions. In designing studies to test for the effects of interventions aiming to alter psychological state, there are concerns that inferences may be hampered by the need to control for non-specific (therapy-related) factors. For this reason, the BRIDGES study was designed with two control groups; a conventional, no-treatment control and an intensive dietary intervention attentionally equivalent to the active mindfulness meditation-based intervention (SRC). Data collection for this three-armed study was completed in April 1999. Results indicate an overall beneficial effect of the SRC that was persistent over time and stronger in women with high baseline emotional distress. Nutrition-related effects were associated exclusively with nutrition-related outcomes, indicating that the SRC effects were specific to that intervention and not just due to non-specific therapy-related factors. There were no effects observed in the usual care control group. Numerous presentations have been made on the findings from this study and the major outcome papers are in various stages of completion, as of this Final Report.

The overall goal in this proposal is to understand the mechanisms by which neurotoxicity and excitotoxicity destroy cells in the substantia nigra. Our hypothesis to test this idea is that c-JUN kinase (JNK), specifically the subtype JNK3, mediates degeneration of substantia nigra neurons after exposure to MPTP or glutamate excitotoxicity. Results in the first year of the project indicate that glutamate agonists at NMDA and kainate receptors increase the biological activity of INK in the basal ganglia (cultured striatal neurons). The subtype JNK3 accounts for most of the JNK activity. Striatal neurons from JNK3 knockout mice lacked most of the biological activity of JNK after glutamate agonist treatment and the size of the JNK responsible for the biological activity corresponded to JNK3. We also observed another, larger protein (complex) with JNK activity. In vivo studies revealed that the MPTP-induced neurotoxicity in wild-type mice was ameliorated in JNK3 knockout mice, but JNK1 and JNK2 offered even greater neuroprotection. Altogether, these year 1 results implicate JNK3 as the principal JNK target of glutamate stimulation in the striatum and JNK's as important mediators of MPTP induced neurotoxicity.

Brown seaweeds are popular foods in Japan, where the incidence of breast cancer is about 1/6 the rate of that reported for American women. Seaweed is an excellent source of fiber, contains iodine, carotenoids, and both mammalian lignans and isoflavones. Seaweed may help to prevent breast cancer by several different mechanisms involving these and other constituents. In a preliminary study of toxicity and efficacy using 5 g/day of seaweed, we found seaweed was well tolerated and was associated with some biological changes in the variables measured. In this study, we will examine the effects of escalating doses I of seaweed supplementation in a group of healthy postmenopausal women with and without breast cancer. In our cross-over design, women will be randomized to either seaweed or placebo first. We will then give doses of seaweed or placebo (3 g/day for 3 weeks, then 6 g/day for 3 weeks), followed by 1 week of 6 g/day seaweed/placebo plus soy (2 mg isoflavone per kg bodyweight). Our primary outcome variables are changes in circulating estrogen levels, thyroid hormones, and urinary excretion of phytoestrogens. Adherence to our program will be measured by monitoring urinary excretion of iodine.

The Breast Research Initiative for Determining Effective Skills for coping with Cancer (BRIDGES) consists of a prospective, randomized intervention trial with sixty women in each of three arms: 1) the UMASS mindfulness meditation-based Stress Reduction and Relaxation Program (SR & RP); 2) a nutrition education program (NEP) developed specifically for BRIDGES; and 3) a usual care control group. The 180 women under age 65 with Stage I or Stage II breast cancer enrolling into this randomized trial are being evaluated for: 1) psychological and behavioral indices of function and coping; 2) Quality of Life (QOL) measures; 3) compliance with the interventions and with medical treatment regimes; and 4) biochemical/immunological measures consisting of cytokines and melatonin. Analyses will be conducted to test hypotheses related to the three specific aims of the study: SR&RP effect on QOL; SR & RP & NEP effects on immune parameters; and durability/decay of intervention-related effects. As of 30 September 1996, the study had closely adhered to the Statement of Work agreed upon at the time the grant was awarded. 178 women were recruited and randomized into the study. Most subjects (90%) who have been randomized and begun the study have been retained. All data have been collected as per protocol stipulations.

Multiple organ dysfunction syndrome (MODS) is most frequent area of late eat in trauma patients and a particular problem for combat casualties where conflict conditions may not allow early evacuation to ICU units of extensive treatment. Consequently, delineating mechanisms for ameliorating posttrauma immunosuppression and overproduction of inflammatory cytokines is a major priority. Only the trauma patient subset with both MOe and T cell dysfunctions develop MODS. This patient subsets' MOe are producing large quantities of deregulated and aberrant TNF(sub alpha), indicated by increased TNF(sub alpha) mRNA stability, failure to shed heutralizing TNF, insensitivity to PGE(sub 2) and TGF(sub beta) downregulation, as well as predominant production of cell associated or mTNF(sub alpha). These aberrant post-injury MOe's ability to activate T cells is also decreased by loss of their IL-12 production and both helper 1 and helper 2 T lymphocyte responses are concomitantly depressed. Dysfunctional T cells fail to appropriately activate or regulate inflammatory MOe allowing exaggerated inflammatory monokines to cause MODS. Aberrant MOe function is detectable as an increase of MOe TNFR (failure to shed the TNFR) and surface expression of mTNF(sub alpha). Aberrant T lymphocyte activity is also rapidly indicated by depressed CD28 and CD3 expression and concomitant upregulation of CD11b expression. Rapid flow cytometric identification of altered MOe and T cell surface receptor/ligand combinations might serve as an easily implementable technique for screening combat casualties. This contract's data have implicated a combination of T lymphocyte and MOe dysfunctions as responsible for the development of MODS. Both a possible means of rapidly identifying combat casualties at risk of MODS and suggestions of future interventive therapy have been developed as a result of this contract

Based on the chemotherapeutic literature and cell culture studies of sulfur mustard (SM) toxicity reported here, we believe that DNA repair mechanisms act as cellular defenses against SM toxicity. Cell culture studies show that survival is increased by the nucleotide excision repair mechanism while biochemical studies show that human as well as bacterial glycosylase enzymes can release two SM- induced DNA adducts: 7-hydroxyethylthioethyl guanine and 3-hydroxyethylthioethyl adenine. The growth of cultured human fibroblasts exposed to 10 uM SM appears to be enhanced in comparison with cells incubated continuously at 37 deg C by subjecting them to a period of hypothermia at 28 deg C before returning them to 37 deg C incubation. Cells held at 28 deg C undergo cell cycle arrest and we believe that this arrest may allow more time for DNA repair before the next round of cell division, thus enhancing survival. Concurrently, we have developed a 32P-postlabeling method that can detect one 7-hydroxy-ethylthioethyl guanine in 10(6) DNA nucleotides in DNA extracted from SM-exposed cells. This method should prove useful in both exposure and DNA repair studies.

Quantitative procedures were developed for testing block-structured models for multi-input nonlinear visual circuits studied with spatiotemporal white noise. A linear-nonlinear (LN) model test index was found to be suitable for classifying cells as simple versus complex. Although simple cells were better modeled as LN systems than complex cells, most simple cells deviated considerably from LN behavior. A nonlinearity of cortical origin would appear to be responsible, possibly activated more strongly by broadband noise than by sinewave grating stimuli. Also, two classes of binocular complex cells were identified. Whereas all binocular complex cells necessarily have a non-zero second-order same-eye interaction kernel, their second-order cross-eye interaction kernel could, it was found, be either non-zero or identically zero. Binocular vision, nonlinear system identification, neural network.

This project is designed to assess the etiology and establish the rapid diagnosis of human viral gastroenteritis. During the contract year for this report, we have continued to demonstrate the involvement of astroviruses in gastroenteritis. In a previously reported Thai study, we have now determined that the clinical symptoms of patients with astrovirus infections were similar to the symptoms of those with rotavirus infection. Additional studies in Guatemala (5,234 samples) and Australia (1,343 samples) have confirmed the importance of astroviruses in gastroenteritis in comparison to other virus infections (rotaviruses and enteric adenoviruses). During this contract year we also developed monoclonal antibodies to calicivirus and we are evaluating these antibodies for use as diagnostic reagents. Ongoing collaborative epidemiological studies have continued to be done with the U.S. military in overseas populations, utilizing the immunodiagnostic techniques that we have developed for gastroenteritis viruses.

The purpose of this contract was to analyze human immune responses to dengue virus infections. To understand the role of immune responses in recovery from dengue virus infection and in the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome, we have analyzed human CD4+ CD8- T cell responses to dengue virus, human CD8+ CD4- T cell responses to dengue viruses, activation of T lymphocytes in vivo in dengue virus infections, dengue virus infection of human cell lines, enhancement of dengue virus infection by antibodies to dengue virus, antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies, production of interferon by dengue virus infected cells, and induction of virus infected monocytes.

During the first reporting period, new equipment was purchased and set up and new software was developed in preparation for electrophysiological experiments to study the neural networks that underly the binocular non-linear filtering properties of cells in the monkey (Macaca fascicularis) visual cortex. This preparatory task was completed. In addition, new methods were developed for using input-output measurements to identify multi-input nonlinear systems. These new mathematical results have been written up and accepted for journal publication and presented at two conferences. In addition, the new system identification methods have been applied in preliminary analyses of previously obtained monocular stimulus-response data. Keywords: Binocular vision; Nonlinear system identification; Neural network.

We have reported that an influenza virus-specific polypeptide produced in E.coli induced influenza virus-subtype-specific memory and secondary H-2 restricted CTL responses in mice. The c13 protein is a hybrid protein of the first 81 amino acids of the NS1 viral non-structural protein and the HA2 subunit of the viral hemagglutinin. We now report results which show that target cells exposed to c13 protein are lysed by virus-immune CTL in a subtype-specific H-2 restricted manner. This suggests that this protein interacts with target cell membranes and is presented on the cell membrane in proper confirmation with H-2 antigens of recognization by the influenza virus-specific CTL. Further, we show that immunization with this molecule results in the induction of virus-specific CTL, which are protective, and this peptide induce CTL without the need for adjuvants. Keywords: Cytotoxic T Lymphocytes, Lymphokines, Epitopes.

We have demonstrated that a conserved portion of the HA2 subunit on the influenza virus hemagglutinin can induce a cytotoxic T lymphocyte response. This is a major development since it raises the possibility that this type of peptide could be used to provide protection that would be cross-reactive among influenza virus strains. The peptide we used was produced in E. coli using recombinant DNA techniques for the expression of segments of influenza viral genome. The molecule which stimulates this H-2 restricted cytotoxic T lymphocyte response is a fusion protein of the HA2 subunit of H1 virus (A/PR/8/34 H1N1), and the induced lymphocytes kill target cells infected with strains of influenza A virus possessing the H1 hemagglutinin irregardless of the years isolated (e.g. 1934, 1978), the results indicate that the HA2 subunit is a candidate for cross- reactive protection because there are substantial published data indicating that influenza virus induced cytotoxic T lymphocytes (Tc) are protective in challenged recipients. Originator supplied keywords include: Lymphokines; and Epitopes.

This paper addresses the etiology, epidemiology, and pathogenesis of human viral gastroenteritis. Effort has been devoted to the development of immunoassays to detect etiologic agents, with the preparation and use of monoclonal antibody reagents where possible. A monoclonal antibody based enzyme immunoassay (EIA) for rotavirus detection was developed, which greatly improves the sensitivity and specificity of diagnosis in neonatal and adult stool specimens over that previously available with polyclonal antibodies. Our older radio-immunoassay procedure for Norwalk virus was adapted to an EIA format, permitting a more convenient and more sensitive assay for epidemiological studies. We demonstrated an immunological relatedness between Norwalk virus and the enteric human calicivirus. Epidemiological studies with collaborating colleagues revealed: the role for Norwalk virus in outbreaks of clam and oyster associated gastroenteritis; and a role for Norwalk virus in 3 to 10 percent of travelers' diarrhea in several countries. Another collaborative study demonstrated the localization of rotavirus by string capsule; this was in contrast to the absence of bacterial enteric pathogens isolated from the same site.

The purpose of this project is to identify, cultivate and characterize ethiologic agents of human viral gastroenteritis, and to study the epidemiology and pathogenesis of infection in order to provide information necessary to attain the goals of prevention and cure of this common syndrome. We have adapted our previously prepared monoclonal antibody reagent against rotavirus to an enzyme immunoassay (EIA) format that is readily adaptable for use in field studies. This monoclonal EIA has greatly improved the sensitivity and specificity of rotavirus EIA detection with neonatal and adult stool specimens. We have also adapted our radioimmunoassay (RIA) for Norwalk virus to an EIA format. Our studies also show that detection of Norwalk antigen by EIA is an more sensitive means for diagnosing infection than serology for some patients. We found a serological relationship between Norwalk virus and the human enteric calicivirus reported by Cubitt. We are also continuing studies to develop monoclonal antibodies against the current noncultivatable Norwalk virus by immunization of mice with extensively purified virus. Keywords: viral diarrhea.

Severe complications are commonly observed in individuals undergoing a secondary dengue infection with a different dengue virus serotype than experienced as the primary infection. Thus, it is important to understand the mechanism(s) of immunologic sensitization which appear to result in more severe subsequent dengue infections. We are developing techniques for detecting viral-specific and non-specific lytic destruction of cells infected with dengue virus. A persistently infected human lymphoblastoid cell line has been developed and used as the target cell system for detecting antibody-dependent cell mediated cytotoxicity (ADCC), and to detect complement-dependent antibody-mediated lysis of dengue virus infected cells. These assays presently, use dengue-type 2 virus infected Raji cells as target cells. Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. Addition of mouse antidengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PMBC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells.

The purpose of this project is to identify, cultivate, and characterize etiologic agents of viral gastroenteritis of man, and to study the epidemiology and pathogenesis of infection in order to provide information necessary to attain the ultimate goals of prevention and cure of this common syndrome. Progress achieved during the present contract year can be summarized as follows. A monoclonal antibody reagent has been developed that is directed against the group specific antigen shared by mammalian rotaviruses; this reagent has then been used to develop two novel rapid diagnostic tests for rotavirus in human stools. These tests provide shortened performance times as well as increased sensitivity and specificity compared to the currently commercially available assay. We are now building upon this experience gained with rotavirus to develop monoclonal antibodies against Norwalk virus, which is to data noncultivatable and fails to produce illness in animal model systems. Approaches are being taken to circumvent logistical problems that have developed in the preparation of monoclonal antibodies to stool derived Norwalk virus. When produced, these antibodies will permit analysis of the bilogy of Norwalk virus, provide a potential diagnostic probe for a possible group-specific antigen common to the noncytopathic Norwalk-like group, and permit recognition of these viruses in nature. In addition, a series of experiments designed to cultivate Norwalk virus in vitro have been undertaken.

We are developing techniques for detecting viral-specific and non-specific lytic destruction of cells infected with dengue virus. This year we developed a persistently infected human lymphoblastoid cell line which has been used as the target cell system for detecting antibody-dependent cell mediated cytotoxicity (ADCC), and to detect complement-dependent antibody mediated lysis of dengue virus infected cells. These assays presently use dengue-type 2 virus infected Raji cells as the target cells. Polyclonal hyperimmune murine sera to dengue types 2 or 4, which is type specific in neutralizing antibody assays, and convalescent human serum contain antidbodies which lyse dengue-2 infected cells in the presence of normal human peripheral blood lymphocytes in this ADCC assay. Hyperimmune polyclonal antisera to dengue 2 virus plus complement lyse dengue 2 infected cells; however, antiserum to dengue 4 virus does not kill dengue 2 infected cells in the presence of complement. This apparent subtype specificity of the complement dependent lytic antibody is unlike the cross-reactive killing of infected target cells in the ADCC assay in which dengue 2 infected cells are killed by hyperimmune antisera to dengue-4 as well as to dengue-2. The lymphocyte responsible for cell lysis in the ADCC assay appears to be T3(-) and OKM 1(+).

An improved method to identify human rotavirus in stool samples was developed through preparation and use of a monoclonal antibody reagent directed against the group specific antigen shared by mammalian rotaviruses. The monoclonal antibody reagent has been adapted to an enzyme-linked immunosorbent assay (EIA), making it available for use in field studies. This monoclonal EIA has considerably improved the sensitivity and specificity of rotavirus EIA detection efforts with neonatal stool samples and adult stool specimens. We are also continuing to develop monoclonal antibodies against the to-date noncultivatable Norwalk virus both by production of human-mouse heterohybridomas and by immunization of mice with extensively purified Norwalk virus. Recently, we began to develop a much needed convenient, quantitative immunoassay for the study of enteric adenoviruses (types 40 and 41)in spite of current difficulties in their recognition in vitro. We reported severe diarrhea produced by rotavirus in adults requiring hospitalization in Thailand, as well as evidence implicating rotavirus as a cause of waterborne gastroenteritis in the U.S. Six U.S. outbreaks of gastroenteritis associated with the consumption of raw and undercooked clams and oysters were shown to be produced by Norwalk virus; one Norwalk virus outbreak was associated with eating food from a salad bar.

The purpose of this project is to assess the medical and epidemiological importance of various etiologic agents of human viral gastroenteritis diagnosis. In addition, we are studying the immunological relationships of gastroenteritis viruses to aid in their classification and diagnosis. Particular attention is being paid to the development and utilization of immunoassays to detect etiologic agents, with the preparation and use of monoclonal antibody reagents where possible. During the contract year for this report, we have prepared and characterized monoclonal antibodies that are reactive with a group antigen shared by all known astroviruses, which are small enteric agents that have been associated with some cases of gastroenteritis. Furthermore we have cultivated in cell culture, visualized, and characterized the Marin County agent of gastroenteritis for the first time, and have shown it to be an astrovirus which also reacts with our astrovirus group specific monoclonal antibodies. Current efforts are underway to develop a sensitive monoclonal antibody based immunoassay for detection of astroviruses in stool samples in order to permit for the first time widespread epidemiological studies on the role of these viruses in gastroenteritis. Keywords: Diarrhea, Adenoviruses.

This project is assessing the etiology and establishing the rapid diagnosis of human viral gastroenteritis. Close attention is being given to the development and utilization of immunoassays to detect various etiologic agents, with the preparation and use of monoclonal antibody reagents were possible. We have succeeded in developing and evaluating an enzyme immunoassay (EIA) test for the direct detection of astrovirus (small,, round diarrhea viruses) in human diarrheal stool samples. This test depends on the use of our previously developed monoclonal antibodies that react with a group antigen shared by all known astroviruses. EIA test to detect astroviruses in stool specimens will now permit us for the first time to assess the role of these viruses in acute gastroenteritis by performing extensive epidemiological studies. Quantitative techniques to be utilized in epidemiological and biological studies of human astroviruses are also being developed through virus plaquing and neutralization techniques. Using these methods, we have established that the Marin County agent of diarrhea is an astrovirus serotype 5, that we have been able to detect directly in stool samples.

The purpose of these studies was to 1) explore the usefulness of a rabbit model to quantitate RBC preservation injury in vivo and to 2) evaluate the importance of RBC-bound C3 in the preservation injury. Several technical aspects relating to homologous transfusions of rabbit RBC were investigated during this study period. These included the method of Cr-51 labeling of the preserved RBC and the comparison of single or double labeling techniques for assessing RBC biability. Preliminary data obtained with double labeling method indicated that 99mTc might be a useful label for fresh autologous RBC. The biological data revealed that ADSOL additive improved the survival of RBC in the initial storage period, but that the viability was not maintained during prolonged storage. Destruction of the complement from plasma of the donor units by heat-treatment (to avoid C3 accumulation to RBC membrane) appeared to have adverse effects on RBC survival. It was thought that heat-treatment produced immune complexes. Additional studies are required to evaluate the role of bound C3 in the preservation injury.

We are describing here in detail a new method to quantitate cell-bound complement in accurate molecular terms. The method is extremely sensitive so that it is possible to quantitate the presence of even ten molecules per cell and it can be utilized to measure complement on various blood cells in-vivo and in-vitro. Utilizing this method we quantitated C3d molecules on fresh RBC and C3b molecules on both stored and quality control RBC (EC43). On the average, 94 C3d molecules and zero C3b molecules were detected on fresh RBC. RBC that had been stored at 4 C for 21 days had acquired about 64 C3b molecules per cell. The number of C3b molecules on heavily sensitized EC43 varied between 2700 to 3000. Other investigators, using radioactive antibodies, detected about 550 C3d molecules on fresh RBC and about 200,000 C3b molecules on EC43. These differences in results apparently reflect methodological bias and could be explained the following way: Since non-monoclonal antibodies were used, they probably consisted of a mixture of antibodies directed to various epitopes on the antigens molecule so that the ratio between radiolabeled antibody and antigen molecules exceeded one. This would favor high results in the radioactive method. In contrast, our results were not calculated on the basis of antibody-antigen ratio but were compared to particle-bound C3 standard. The new immunochemical method will be applied for quantitation of C3 on various populations of stored RBC to determine whether RBC aged in-vivo are more susceptible to complement-induced storage lesion than young RBC. (Author)