Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer. Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.

Graduation date: 1993

Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

The usual approach to the study of signaling pathways in biological systems is to assume that high numbers of cells and of perfectly mixed molecules within cells are involved. To study the temporal evolution of the system averaged over the cell population, ordinary differential equations are usually used. However, this approach has been shown to be inadequate if few copies of molecules and/or cells are present. In such situation, a stochastic or a hybrid stochastic/deterministic approach needs to be used. Moreover, considering a perfectly mixed system in cases where spatial effects are present can be an over-simplifying assumption. This can be corrected by adding diffusion terms to the ordinary differential equations describing chemical reactions and proliferation kinetics. However, there exist cases in which both stochastic and spatial effects have to be considered. We study the relevance of differential equations, stochastic Gillespie algorithm, and deterministic and stochastic reaction-diffusion models for the study of important biological processes, such as viral infection and early carcinogenesis. To that end we have developed two optimized libraries of C functions for R (r-project.org) to simulate biological systems using Petri Nets, in a pure deterministic, pure stochastic, or hybrid deterministic/stochastic fashion, with and without spatial effects. We discuss our findings in the terms of specific biological systems including signaling in innate immune response, early carcinogenesis and spatial spread of viral infection.

Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of liver carcinogenesis. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from conditioned medium of Huh-7 hepatoma cell line. HDGF has growth stimulating activity for various types of cells. Recent evidence indicates that HDGF upregulation is associated with poor survival outcome and tumor progression in HCC, non-small cell lung carcinoma and melanoma. However, the exact function and molecular mechanism of HDGF overexpression during HCC progression remain largely unknown. In the first project (Chapter 2) of this thesis study, we started with characterizing in HDGF release and response to exogenous HDGF between benign HepG2 and malignant SK-Hep-1 hepatoma cells. It was found that serum deprivation significantly stimulated the HDGF secretion in SK-Hep-1 cells but not HepG2 cells. Interestingly, SK-Hep-1 cells did not increase the secretion of vascular endothelial growth factor (VEGF), a potent angiogenic factor, during serum deprivation. Besides, SK-Hep-1 cells were more responsive to the growth- and migration-promoting effect of exogenous HDGF. We also validated the angiogenic functions of recombinant HDGF protein in vitro and in vivo. In the second project (Chapter 3), we investigated the influence of cellular HDGF level on the neoplastic potential of hepatoma cells. Adenovirus vectors encoding HDGF, Ad-HDGF, and antisense HDGF, Ad-HDGF (-), were generated to modulate the cellular HDGF levels in SK-Hep-1 cells. Adenovirus-mediated HDGF gene delivery increased the HDGF expression and release, and stimulated the proliferation, migration and anchorage-independent growth of SK-Hep-1 cells. In contrast, infection with Ad-HDGF (-) reduced the HDGF expression and secretion, and attenuated the oncogenic behaviors of SK-Hep-1 cells. Implanting HDGF-overexpressing SK-Hep-1 cells led to the accelerated growth of xenografted hepatoma in SCID mice while implantation of HDGF-downregulated SK-Hep-1 cells caused retarded tumor growth. Histological analysis revealed the increased proliferation and neovascularization in HDGF-overexpressing tumors. This could be attributed to elevated VEGF expression and activation of the nuclear factor kappa B (NF£eB) activities by HDGF upregulation in SK-Hep-1 cells. In the third project (Chapter 4), we delineated the mechanism underlying HDGF-induced VEGF secretion and activation of NFB pathway in SK-Hep-1 cells. Adding recombinant HDGF protein enhanced the VEGF release by SK-Hep-1 cells particularly during serum starvation. This was associated with a concomitant increment in VEGF protein and mRNA levels in SK-Hep-1 cells. Like many mitogens, HDGF increased the production of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide in a dose-dependent manner. Pretreatment with antioxidants abolished the HDGF-induced VEGF secretion. NF£eB is a pivotal transcription factor for regulation of pro-inflammatory cytokines and genes such as VEGF and cycloxygenase¡V2 (COX-2). Application of HDGF stimulated NF£eB-driven luciferase activities. This was correlated with a dose- and time-depedent increment of NF£eB (p65) by HDGF. HDGF treatment also elevated the COX-2 protein levels and activities in SK-Hep-1 cells. In addition, blockade of COX-2 by NS-398 attenuated the HDGF-induced VEGF secretion, suggesting the involvement of COX-2. Finally, it was found that HDGF stimulated the phosphorylation of Akt, Erk1/2, and p38 MAPK. Inhibition of Akt by LY294002 also diminished the HDGF-induced VEGF secretion. These studies suggest that HDGF induces oxidative stress to activate NF£eB/COX-2/Akt pathway, thereby stimulating VEGF expression and release. In summary, this thesis study brings functional and mechanistic insights on how aberrant HDGF expression contributes to angiogenesis and tumorigenesis during liver carcinogenesis.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains viii, 124 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 1987.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 210 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 154-185).

Energy balance refers to the relationship between energy intake and energy expenditure. Epidemiological studies have established a clear association between energy balance and cancer, however the underlying mechanisms are unclear. The objective of the current study was to evaluate the impact of caloric consumption on epithelial carcinogenesis and identify potential mechanisms of inhibition or enhancement. Using ICR female mice, we demonstrated that positive energy balance enhanced, while negative energy balance inhibited susceptibility to multistage carcinogenesis in mouse skin. We next evaluated diet-induced changes in the epidermal proliferative response. Calorie restriction (CR) significantly reduced epidermal hyperproliferation, in the presence and absence of tumor promotion, as compared to diet-induced obesity (DIO). Additional studies were conducted to determine the impact of dietary manipulation on TPA-induced growth factor signaling. CR reduced, while DIO increased insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation, which subsequently modulated signaling downstream to Akt and mTOR. These diet-induced changes in growth factor signaling were confirmed under steady-state conditions in multiple epithelial tissues (i.e., skin, liver and dorsolateral prostate) in multiple mouse strains (FVB/N, C57BL/6 and ICR). Further analyses demonstrated that caloric consumption directly correlated with levels of cell cycle progression related proteins and inversely correlated with levels of cell cycle inhibitory proteins. Genetic reduction of circulating IGF-1, liver IGF-1 deficient (LID) mouse model, inhibited two-stage skin carcinogenesis, reduced epidermal hyperproliferation and attenuated IGF-1R and EGFR growth factor signaling during tumor promotion, similar to CR, suggesting a potential for IGF-1R and EGFR crosstalk. Further studies, demonstrated that IGF-1 induced EGFR activation in cultured mouse keratinocytes, possibly due to IGF-1R and EGFR heterodimerization or IGF-1 induced changes in EGFR mRNA expression. In vivo, CR reduced, while DIO increased IGF-1R and EGFR association during tumor promotion. Furthermore, CR attenuated EGFR ligand mRNA expression both in the presence and absence of TPA treatment. Collectively, these findings suggest that dietary energy balance modulates epithelial carcinogenesis, at least in part due to diet-induced changes in levels of circulating IGF-1, which then modulate IGF-1R and EGFR crosstalk and downstream signaling to cell cycle related proteins, subsequently altering epidermal hyperproliferation. / text