Increased tumor proliferation and genomic instability without decreased apoptosis in MMTV-ras mice deficient in p53.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10(5 )deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 +/- 0.002 for liver to 0.015 +/- 0.003 for brain. We observed a significant increase (10-fold) in oxo8dG in nDNA isolated from liver tissue after 2 Gy of gamma-irradiation when NaI was used to isolate DNA. The turnover of oxo8dG in nDNA was rapid, e.g. disappearance of oxo8dG in the mouse liver in vivo after gamma-irradiation had a half-life of 11 min. The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues. Thus, our results showed that the steady-state levels of oxo8dG in mouse tissues range from 180 to 360 lesions in the nuclear genome and from one to two lesions in 100 mitochondrial genomes.

Differential regulation of cell cycle characteristics and apoptosis in MMTV-myc and MMTV-ras mouse mammary tumors.

We have used the MMTV-myc and MMTV-ras transgenic mouse mammary tumor models (T. A. Stewart et al., Cell, 38: 627-637, 1984, and E. Sinn et al., Cell, 49: 465-475, 1987) to evaluate how the c-myc and v-Ha-ras oncogenes influence tumor growth characteristics in vivo. MMTV-myc tumors had much higher levels of spontaneous apoptosis than MMTV-ras tumors, whereas intermediate levels were observed in MMTV-myc/ras tumors. Significant differences in cell cycle characteristics were also observed in tumors from mice of the three genotypes. Tumors from MMTV-myc mice had lower G1 and higher S-phase fractions than MMTV-ras tumors, with intermediate values again observed in the MMTV-myc/ras tumors. Despite these differences, however, tumor growth rates for the different groups were similar. These findings highlight the importance of the balance between cell cycle regulation and cell death in determining the kinetics of tumor growth and indicate that distinct oncogenes can have a profound influence on that balance.

Prognostic value of Ki67 in localized prostate carcinoma: a multi-institutional study of >1000 prostatectomies.

BACKGROUND: Expanding interest in and use of active surveillance for early state prostate cancer (PC) has increased need for prognostic biomarkers. Using a multi-institutional tissue microarray resource including over 1000 radical prostatectomy samples, we sought to correlate Ki67 expression captured by an automated image analysis system with clinicopathological features and validate its utility as a clinical grade test in predicting cancer-specific outcomes. METHODS: After immunostaining, the Ki67 proliferation index (PI) of tumor areas of each core (three cancer cores/case) was analyzed using a nuclear quantification algorithm (Aperio). We assessed whether Ki67 PI was associated with clinicopathological factors and recurrence-free survival (RFS) including biochemical recurrence, metastasis or PC death (7-year median follow-up). RESULTS: In 1004 PCs (∼4000 tissue cores) Ki67 PI showed significantly higher inter-tumor (0.68) than intra-tumor variation (0.39). Ki67 PI was associated with stage (P<0.0001), seminal vesicle invasion (SVI, P=0.02), extracapsular extension (ECE, P<0.0001) and Gleason score (GS, P<0.0001). Ki67 PI as a continuous variable significantly correlated with recurrence-free, overall and disease-specific survival by multivariable Cox proportional hazard model (hazards ratio (HR)=1.04-1.1, P=0.02-0.0008). High Ki67 score (defined as ⩾5%) was significantly associated with worse RFS (HR=1.47, P=0.0007) and worse overall survival (HR=2.03, P=0.03). CONCLUSIONS: In localized PC treated by radical prostatectomy, higher Ki67 PI assessed using a clinical grade automated algorithm is strongly associated with a higher GS, stage, SVI and ECE and greater probability of recurrence.

Genetic determinants of response to chemotherapy in transgenic mouse mammary and salivary tumors.

Several transgenic mouse tumor models were utilized to explore how specific genetic alterations affect the tumor cell response to chemotherapeutic agents in vivo. Specifically, MMTV-ras transgenic mice were interbred to p53 knock-out mice to create a model for assessing the role of p53 in chemotherapeutic responses. In addition, MMTV-ras tumors were compared to MMTV-myc and MMTV-ras/myc tumors. Mice of each genotype reproducibly develop mammary and/or salivary tumors, but tumor growth dynamics vary considerably between genotypes. MMTV-ras/p53-/- tumors exhibit higher S phase fractions than MMTV-ras/p53+/+ tumors, although both tumor types display very low apoptosis levels. In contrast, MMTV-myc tumors exhibit both high S phase fractions and spontaneous apoptosis levels. Tumor-bearing mice of each genotype were treated with either doxorubicin or paclitaxel, and effects on overall tumor growth, cell cycle distribution and apoptosis were evaluated. Surprisingly, neither agent efficiently induced apoptosis in any of the tumor models, including those with wildtype p53. Rather, tumor responses were mediated primarily by changes in cell cycle distribution. However, the spontaneous apoptosis levels did serve as a predictor of tumor growth response, in that only those tumors with high pretreatment apoptosis levels underwent significant regression following treatment with either agent.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells.

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Disruption of functions of wild-type p53 by hetero-oligomerization.

We report that a p53 segment (p53 del 1-293) containing the oligomerization domain interferes with the functions of wild-type p53. Wild-type p53 inhibits transcription mediated by human cytomegalovirus (CMV) immediate-early promoter significantly; however, co-expression of p53 del 1-293 drastically reduces this repression. We show that wild-type p53 forms hetero-oligomers with p53 del 1-293 suggesting that the hetero-oligomers are defective in repressing the CMV promoter. A synthetic promoter with p53-binding sites is transactivated significantly by wild-type p53. However, co-expression of p53 del 1-293 drastically reduces this activation. At a high concentration, a deletion mutant of wild-type p53 (del 393-327) defective in oligomerization transactivates efficiently a promoter with synthetic p53-binding sites. This transactivation remains unaffected by co-expression of p53 del 1-293. p53 del 393-327 also fails to hetero-oligomerize with p53 del 1-293 indicating that hetero-oligomerization is necessary for disruption of wild-type p53-mediated transactivation. Immunostaining experiments show that hetero-oligomerization does not lead to changes in localization of nuclear p53 demonstrating that delocalization of p53 is not the reason for inactivation. We also show that co-expression of p53 del 1-293 significantly reduces the G1/S arrest by wild-type p53 suggesting that a proper oligomeric form is necessary for wild-type p53-mediated cell cycle arrest. Thus, our work shows that hetero-oligomerization disrupts wild-type p53's biological functions and suggests a mechanism by which p53 mutants may disrupt functions of wild-type p53.

Promise and challenge: Markers of prostate cancer detection, diagnosis and prognosis.

Approximately 1 man in 6 will be diagnosed with prostate cancer during his life lifetime, and over 200,000 men in the U.S. are diagnosed with prostate cancer annually. Since the widespread adoption of PSA testing, about 60-70% of men at risk in the U.S. have had a blood test for prostate cancer. With this, prostate cancer death rates have decreased, yet only slightly. Thirty thousand men still die each year from this disease. PSA testing fails to identify a small but significant proportion of aggressive cancers, and only about 30% of men with a "positive" PSA have a positive biopsy. Additionally, of men who are treated for prostate cancer, about 25% require additional treatment, presumably due to disease recurrence. Also of concern is the growing evidence that there are some prostate cancers for which treatment may not be necessary. Very long-term studies from the U.S. and Europe, following men with prostate cancer have found that some tumors do not progress over time. In these individuals, prostate cancer treatment is unnecessary and harmful as these men do not benefit from treatment but will be at risk of treatment-related side effects and complications. They suggest a fundamental problem with prostate cancer: it is not possible, at this time, to predict the natural history of the disease. It is for these reasons that the most important challenge in prostate cancer today is the inability to predict the behavior of an individual tumor in an individual patient. Here we review issues related to performance and validation of biomarkers with a focus on "doing no harm", and bearing in mind that it is the ultimate goal of early detection to save lives. Improved diagnostic and prognostic biomarkers are needed for prostate cancer, and the use of these markers should ultimately translate into increased life span and quality of life. The ultimate goal would be to not only have accurate biomarkers suitable for early diagnosis, but also biomarkers that identify men at greatest risk of developing aggressive disease. Technology has been brought to bear on this problem, and the major approaches are genomics, expression analysis, and proteomics. Proteomics and DNA methylation assays may soon be used in sensitive and specific diagnostic testing of serum and tissues for cancer. Expression arrays may be used to establish both a more specific diagnosis and prognosis for a particular tumor. The proteome is only beginning to be understood, and alternative splicing and post-translational modifications of proteins such as glycosylation and phosphorylation are challenging areas of study. Finally, risk assessment and prognosis are being pursued through analysis of genomic polymorphisms (single nucleotide polymorphisms, SNPs). This huge task is only beginning, and requires the combined expertise of molecular epidemiologists, oncologists, surgeons, pathologists, and basic scientists.

Modulation of antioxidant enzymes and programmed cell death by n-3 fatty acids.

Studies from our laboratory indicate that n-3 (fish oil, FO) lipids at 10% (w/w) in a nutritionally adequate, semipurified diet, and supplemented with equal levels of antioxidants, extended the life span of lupus-prone (NZB/NZW)F1 (B/W) female mice as compared to n-6 (corn oil, CO) lipids. The early rise of autoimmune disease in CO-fed mice was closely linked to the loss of T-cell function. Both IL-2 production and IL-2 receptor expression were reduced due to the loss of naive T-cells and a rise in memory T-cells. Proliferative response to both mitogens and superantigens (staphylococcal enterotoxins A and B) was higher in FO-fed 6.5-mon-old mice. These changes paralleled decreased PGE2 production by splenic cells from FO-fed mice. Analysis of mRNA expression in different organs revealed differential effects of dietary lipids. In FO-fed mice, transforming growth factor beta 1 (TGF beta 1) expression was decreased in kidneys, but splenic tissues had higher expression of TGF beta mRNA. As TGF beta promotes programmed cell death (PCD), we studied the effects of CO and FO on PCD rates in lymphocytes. Both propidium iodide staining and DNA fragmentation were elevated in lymphocytes of FO-fed mice when compared to CO-fed mice of similar age. Also, increased PCD correlated closely with increased Fas gene expression. Thus, in addition to various other antiinflammatory effects, dietary FO appears to increase PCD and prevent accumulation of self-reactive immune cells in lymphoid organs. Further studies are required to dissect the pro- and antiinflammatory mechanisms associated with dietary n-3 and n-6 lipids in modulating autoimmune disorders or malignancy during aging.

Molecular pathology of prostate cancer.

This chapter includes discussion of the molecular pathology of tissue, blood, urine, and expressed prostatic secretions. Because we are unable to reliably image the disease in vivo, a 12 core method that oversamples the peripheral zone is widely used. This generates large numbers of cores that need to be carefully processed and sampled. In spite of the large number of tissue cores, the amount of tumor available for study is often quite limited. This is a particular challenge for research, as new biomarker assays will need to preserve tissue architecture intact for histopathology. Methods of processing and reporting pathology are discussed. With the exception of ductal variants, recognized subtypes of prostate cancer are largely confined to research applications, and most prostate cancers are acinar. Biomarker discovery in urine and expressed prostatic secretions would be useful since these are readily obtained and are proximate fluids. The well-known challenges of biomarker discovery in blood and urine are referenced and discussed. Mediators of carcinogenesis can serve as biomarkers as exemplified by mutations in PTEN and TMPRSS2:ERG fusion. The use of proteomics in biomarker discovery with an emphasis on imaging mass spectroscopy of tissues is discussed. Small RNAs are of great interest, however, their usefulness as biomarkers in clinical decision making remains the subject of ongoing research. The chapter concludes with an overview of blood biomarkers such as circulating nucleic acids and tumor cells and bound/free isoforms of prostate specific antigen (PSA).

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line.

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

Differential alterations in 5alpha-reductase type 1 and type 2 levels during development and progression of prostate cancer.

BACKGROUND: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development. METHODS: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases. RESULTS: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively. CONCLUSIONS: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa.

Lipid alterations in LLC-PK1 cells exposed to mercuric chloride.

We have studied the effects of HgCl2 on the lipids of LLC-PK1 (pig kidney) epithelial cells. Our results show that treatment of cells with HgCl2 caused a rapid accumulation of unesterified fatty acids (particularly arachidonic acid) and lysophospholipids. A 27-fold increase in unesterified arachidonic acid and a 17-fold increase in lysophosphatidylethanolamine (LPE) was accompanied by a 26% decline in the mass of phosphatidylethanolamine as determined by gas chromatography and lipid phosphorus assay. Similar changes were seen following HgCl2 treatment of cells whose lipids were labelled with 14C stearic acid, 3H arachidonic acid, or 14C acetate, but the radiolabelling techniques also identified an increased content of label in lysophosphatidylcholine (LPC) and a corresponding decrease in phosphatidylcholine. These alterations were accompanied by the formation of blebs on the plasma membrane and irreversible injury as indicated by electron microscopy. The possible role of unesterified fatty acids in the pathogenesis of injury was studied by adding fatty acids to the cells. The addition of unsaturated fatty acids (oleic, linoleic, or arachidonic acids) to the cells caused plasma membrane blebbing and loss of viability. Similarly, the addition of LPC or LPE to the cells resulted in cell death; however, plasma membrane blebbing did not result.

Effects of calorie restriction and ω-3 dietary fat on aging in short-and long-lived rodents.

Aging is accompanied by a steady increase in the incidence of spontaneous tumors and a decline in immune function. Calorie restriction (CR) or supplementation with ω-3 fats prolongs life span, suppresses tumorigenesis, and ameliorates immune function in a variety of experimental models. We suggest that decreased oxidant stress and upregulation of apoptosis mediate the effects of calorie restriction on immunity and longevity. CR prolongs life span in several animal models and our studies have examined the effects of CR on the immune system and on tumorigenesis. CR maintains naive T cells, prevents the rise in "double-negative" T cells, maintains lymphocyte responsiveness to mitogens, and preserves Dexamethasone induced apoptosis in spleen cells of MRL/Ipr mice. CR also modulates the expression of inflammatory mediators and cytokines. CR decreases the Sjögren's syndrome-like chronic inflammation of salivary glands of B/W animals while increasing expression of the immunosuppressive cytokine TGFß1 and decreasing expression of the pro-inflammatory cytokines IL-6 and TNFα. The autoimmune disease in the B/W mouse also affects the kidneys, and we find that renal expression of platelet derived growth factor-A, (PDGF-A) and thrombin receptor are decreased in CR animals. Similarly, CR decreases the expression and localization of plasminogen activator inhibitor type 1 in glomeruli of B/W animals. CR also modulates expression and function of androgen receptors and the binding of insulin to liver nuclei. Finally, CR suppresses the development of breast tumors in the Ras oncomouse. These effects of calorie restriction are paralleled in short-lived B/W animals fed diets supplemented with ω-3 fatty acids. Omega-3 fatty acids induce the expression of hepatic antioxidant enzymes, and enhance apoptosis in lymphocytes of B/W animals.

Two-chain urokinase, receptor, and type 1 inhibitor in cultured human mesangial cells.

Urokinase-type plasminogen activator (u-PA), its receptor (u-PAR), and type 1 inhibitor (PAI-1) in cultured human mesangial cells were investigated. Treatment with phospholipase C (PLC) released plasminogen activators [with relative mol wt (M(r)) of 55,000 and 100,000] and u-PAR into the culture medium. By Western blot, both u-PA and PAI-1 were present in the M(r) 100,000 band. Since PAI-1 binds only active, two-chain u-PA (tcu-PA), formation of the M(r) 100,000 band reflects conversion of the single-chain, proenzyme form of u-PA (scu-PA) to tcu-PA. Immunofluorescence staining of whole cells demonstrated the presence of PAI-1, u-PA, and u-PAR. Immunofluorescence staining and Western blot analysis showed enrichment of PAI-1, u-PA, and u-PAR in a preparation of substratum-attached extracellular matrix and membrane proteins termed adhesion plaques. Using a chromogenic assay, we found that PAI-1 expression in adhesion plaques exceeded that of u-PA. We conclude that cultured human mesangial cells produce receptor-bound u-PA/PAI-1 complexes localized to adhesion plaques.

Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

The effects of dietary lipids on gene expression and apoptosis.

The beneficial effects of dietary FO with respect to autoimmune disease, CVD and some types of cancer are well established. Studies conducted over the last 10-15 years have established the potent effects of FO on gene expression in the previously mentioned diseases. The effects of dietary FO appear to be selective in nature, with the expression of individual genes simultaneously being increased, decreased or completely unaffected. In order to elucidate the molecular mechanism(s) involved, recent studies have focused on analysing the effects of the long-chain polyunsaturated n-3 fatty acids EPA and DHA which are highly enriched in FO and thought to be the primary mediators of its biological activity. Indeed, it has been found that EPA and DHA appear to both directly and indirectly modulate gene expression in vivo, depending on the gene examined. The direct effects of EPA and DHA are most probably mediated by their ability to bind to positive and/or negative regulatory transcription factors, while the indirect effects appear to be mediated through alterations in the generation of intracellular lipid second messengers (e.g. diacylglycerol and ceramide). Future studies need to be focused on further elucidation of the inter- and intracellular signalling events mediated by dietary n-3 fatty acids. Understanding the molecular mechanism(s) modified by dietary FO will ultimately lead to improved dietary strategies to aid in the prevention of autoimmune disease, CVD and/or certain types of cancer.