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Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed May 20, 2008). PDF text: xii, 238 p. : ill. ; 11 Mb. UMI publication number: AAT 3284223. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Includes bibliographical references. Also available online.

Mechanistic insights on molecular and cellular mechanisms whereby KSHV induces Kaposi?s sarcoma (KS) are key for our understanding of KS tumors and for the development of new therapies. We have previously developed an animal model for KSHV induced KS using murine bone marrow cells transfected with a KSHVBac36. We found that although these cells lacked attributes of transformed cells in vitro, they were able to cause KS-like tumors in vivo. In vivo tumorigenesis correlated with upregulation of both KSHV lytic genes and host angiogenesis suggesting that that cues provided from the microenvironment played a major role in regulating viral and host gene expression related with KSHV-induced tumorigenesis. Our goal thus, was to identify these molecular cues regulating pathogenic gene expression in KSHV infected cells in vivo. An important difference between cells kept in vitro versus in vivo is the lack of environmental extracellular matrix (ECM) signals. Therefore the mECK36 cells were cultured in vitro in matrigel, a basement membrane preparation rich in ECM proteins and its individual components, to discern the effect of host signaling by the ECM on KSHV infected cells. Investigation of gene expression through Real Time RT-PCR identified several viral and host genes associated with tumorigenesis such as KSHV vGPCR and angiogenesis associated VEGF and EGF- receptors were upregulated in response to this environment. Further analysis of the molecular activity of the cell indicated the change in transcription was due to the activation of integrin signaling, as assessed by phosphorylation of the Focal Adhesion Kinase (FAK) protein. Our results show that integrin signaling occurring in vivo through interaction with ECM serves to enhance the pathogenic viral and host gene expression of KSHV infected cells and that EGFR upregulation can be correlated with these conditions. These results points to the integrin signaling pathway or the EGF-Receptor as promising targets for therapy and prevention of KS tumors.

Predictors of response of AIDS-associated Kaposi-Sarcoma to standard chemotherapy Overview: Kaposi Sarcoma is the most common HIV-associated cancer. Its etiology and pathogenesis is not fully understood. Little is known about what predicts prognosis, survival and therapeutic response in HIV-KS. In South Africa given the high seroprevalence rates of HIV-l and human herpes virus 8 (HHV 8), Kaposi's sarcoma is a significant problem. The majority of patients have been treated solely with palliation due to the poor outcome associated with a diagnosis of HIV-KS, more so in the absence of highly active antiretroviral therapy (HAART). Since the national ARV rollout programme and the availability and accessibility of HAART to all patients with a diagnosis of HIV-KS, a new strategy has to be established to enable adequate patient selection for chemotherapy. There have been a few published studies addressing the predictors of response to chemotherapy in the first world. However, this is the first study of these factors in HIV-l infected African patients with Kaposi's sarcoma. Aim: To identify and assess the potential value of several parameters predictive of outcome, survival and therapeutic response in HIV- infected patients with KS. Clinical, hematological, biochemical, immunological and virological variables were evaluated. Methods: We collected data from 25 patients with AIDS-KS who were enrolled in a phase III randomized controlled trial comparing HAART alone with the combination of HAART and chemotherapy. All patients were from the combination therapy arm. The following variables were evaluated as predictors of prognosis and therapeutic response: age, gender, ethnic origin, Haemoglobin (Hb), white blood cells (WBCs), lymphocytes, neutrophils, platelets, S.albumin, ALP, GGT, CD4 count, HIV viral load. These variables were assessed in patients at baseline and month 6 of therapy. Patients were staged into good risk and poor risk according to the AIDS clinical trial group (ACTG) criteria. The outcomes assessed were response to treatment and mortality. Results: A total of 25 patients participated to the study. Of these 16(64%) were males and 9(36%) were females, with male: female ratio of 2.7:1. Median age was 34 years (24-47); all patients were of Black African origin. Of the 21 patients, 15 (71.4%) were of good prognosis and 6(28.6%) were of poor prognosis. At baseline the median values of the different variables were as follows: Hb 10.9 g/dl, WBCs 5.95x109/L, lymphocytes 1.7 x109/L, neutrophils 3 x10 9 /L, platelets 272 x10 9 /L, S.albumin 30 gil, total protein 88 gil, ALP 64 U/L, and GTT 21 U/L, CD4 count was 255 cells/mm 3 , HIV-RNA viral load was 42000( 4.610gs). At month 6, 22 patients remained alive, their median values were: Hb 12.2 g/dl, WBCs 4.65 x109/L, lymphocytes 1.5 x109/L, neutrophils 3 x10 9 /L, platelets 301 x109/L, S.albumin 36.5 gil, total protein 84.5 gil, ALP 78.5 U/L, GTT 44.5 U/L, CD4 count 288 cells/mm3 , HIV-RNA viral load was 50500( 4.6910gs). The baseline median CD4 and HIV-RNA viral load counts for the 3 patients who died before month 6 were 47 cells/mm3 and 31000(4.610gs); respectively. Response to therapy was evaluated in 21(84%) patients as 4(16%) patients were missing, of the 21 patients 3 (14.3%) had complete response and 18(85.7%) had partial response. With respect to sex 2(14.3%) males had complete response and 12(85.7%) had partial response, 1(14.3%) female had complete response and 6 (85.7%) had partial response. Non-parametric statistics were used because of the small sample size and the skewness of the data. Variables were described using medians and ranges, and compared between two independent groups using Mann-Whitney tests. Baseline and month 6 comparisons were done using Wilcoxon signed ranks tests. Receiver Operating Characteristic (ROC) curves were used to analyze cut points to optimize sensitivity and specificity of a quantitative variable for a dichotomous outcome. Discussion In the univariate analysis age and sex didn't influence prognosis and therapeutic response, the influence of ethnic origin couldn't be assessed as all patients were of the same ethnic origin. Baseline WBCs (P= 0.004) and lymphocytes (P=0.026) were significantly associated with complete response. Higher values of GGT (p=O.OOl); ALP (P=0.006) were associated with more deaths. Baseline CD4 count and HIV viral load were not of predictive value, lthough change CD4 (P=002) and VL (p=.OOO) over time was significant and most likely attributed to response to therapy. 90.9 % of patients reached undetectable HIV-l Viral loads at month 6. CONCLUSION: Neither CD4 count nor HIV viral load at baseline predicted prognosis or survival; however there was a borderline significance of CD4 (P=0.058) towards a better survival. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2006.

Acute oncogenic stress can activate autophagy and facilitate permanent arrest of the cell cycle through a failsafe mechanism known as oncogene-induced senescence (OIS). Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS) and has been reported to encode oncoproteins within its latency program that engage host autophagy and OIS pathways; however, the mechanisms by which KSHV oncoproteins promote KS tumorigenesis remain unclear. Here, I demonstrate that ectopic expression of the latent KSHV protein viral cyclin (v-cyclin) deregulates the cell cycle, induces DNA-damage responses (DDRs) and promotes OIS through an autophagy-dependent mechanism. During latency, v-cyclin is co-expressed from a single transcript with a viral homolog of FLICE-inhibitory protein (v-FLIP) that blocks autophagy by binding and inhibiting Atg3. Co-expression of v-FLIP has no effect on DDRs, but efficiently blocks v-cyclin-induced autophagy and senescence. Remarkably, suppression of v-FLIP function during KSHV latency, through specific inhibitory peptides, rescues host cell autophagy and induces senescence of infected cells. Together, these data reveal a coordinated viral gene-expression program that subverts autophagy, impairs senescence, and facilitates the proliferation of KSHV-infected cells.

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 5 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Medicine. Includes list of published articles and presentations. Includes bibliographical references. Also available in print form.