Cigarette smoke, DNA damage and repair in human cervical and vaginal cells in vitro and ex vivo.

Among gynecological malignancies, carcinoma of the cervix is the leading cancer type worldwide. Conversely, vaginal tumors account for an incidence of approximately 1-2% of lower genital tract malignancies. Interestingly, the major shared risk factor among women with cervical and vaginal tumors is infection with cancer-associated human papillomaviruses (HPVs). In fact, HPV infection is the necessary causative factor in the development and progression of cervical and vaginal neoplasia in the majority of cases. Epidemiological studies support that cervical and vaginal pre-cancerous lesions that are initiated by HPV infection generally regress in the absence of known risk factors such as cigarette smoking. Further, female smokers have two times higher risk of developing cervical cancer from premalignant lesions than non-smokers, although the association between cigarette smoking and vaginal tumors has been controversial. Cigarette smoking exerts its cytogenetic effects in various forms such as DNA strand breaks and oxidative DNA damage. However, previous studies on cervical and vaginal abnormalities failed to investigate the effects of cigarette smoking on cervical and vaginal cells from a biological standpoint. Therefore, the goal of this work was to investigate cigarette smoke-induced DNA strand breaks, oxidative damage and DNA repair in human cervical cancer cells (in vitro studies, Chapters II and III); we also examined DNA strand breaks in human vaginal cells in light of cigarette smoking and HPV status (an ex vivo study, Chapter IV). It was determined that cigarette smoke induces both DNA single- (SSBs) and double-strand breaks (DSBs) that are highly persistent, in addition to oxidative DNA damage, which is more pronounced in HPV infected human cervical cancer cells. Though, CSC-induced oxidative DNA damage was eventually removed in the cells, the possibility of error-prone DNA repair, however, cannot be ruled out. Lastly, our data derived from the ex-vivo study clearly showed an inverse relationship between the interaction of cigarette smoking and HPV infection in the induction of DNA strand breaks in vaginal cells of the studied population, regardless of age. Further, we have clearly showed that the extent of DNA DSBs increases as a function of age, which corroborates with the fact that vaginal cancers occur at a much older age, as compared to cervical cancers. In summary, the findings of this research project shed light on the role of cigarette smoke in cervical/vaginal carcinogenesis. Moreover, comet assay might serve as an early biomarker in identifying susceptible populations that are at risk for developing cervical/vaginal abnormalities

Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy

Lentivirus-mediated delivery of transcription factors to CPCs.

<p>A, Untranduced or mCherry virus-transduced CPCs were imaged at 4 days post-transduction. Fluorescence of mCherry protein is visible in red. DAPI staining of nuclei was pseudo-colored in green. B, Cells transduced with virus expressing mCherry (control), 3xFLAG-tagged Gata4, MEF2C, TBX5 or BAF60C, or V5-tagged NKX2.5 were assayed by Western blot using antibody against each TF. C, Cells transduced with virus expressing 3xFLAG-tagged Gata4, MEF2C, TBX5 or BAF60C, or V5-tagged NKX2.5 were stained for the indicated epitope (i.e., FLAG or V5) which is shown in monochrome. DAPI images are shown in lower panels. Note that exogenous, epitope-tagged transcription factors localize to the nucleus as expected.</p

Effect of individual transcription factors on expression of cardiac differentiation markers in human CPCs.

<p>Transcription factors (<i>Gata4</i> [G], <i>MEF2C</i> [M], <i>NKX2</i>.<i>5</i> [N], and <i>TBX5</i> [T]) were introduced to c-kit+ and lin- human CPCs via lentivirus-mediated gene delivery. Cells were then cultured for 1 or 2 weeks. At each time point, cells were harvested, and relative changes in mRNA levels of indicated genes were measured using quantitative RT-PCR. The level of indicated transcript in each group was compared to that of mCherry (mCh)-expressing control group, and is expressed as a relative fold change. For each condition, <i>n</i> = 4. Bar graphs show mean ± SEM. *, <i>p</i> < 0.05. †, not detected.</p

Gata4-induced changes in gene expression and morphology.

<p>A, Representative phase-contrast images of mCherry (control) and <i>Gata4</i>-overexpressing CPCs are shown (upper panels). The indicated cells were stained with Alexa Fluor 488-conjugated phalloidin after 2 weeks of culture to visualize actin cytoskeleton (lower panels). Note that control cells remain spindle-shaped and elongated, while cells overexpressing Gata4 appear polygonal in their morphology. B, CPCs were transduced as in Panel A and stained for the indicated markers after 2 weeks of culture. BNP staining within <i>Gata4</i>-expressing cells are indicated by the arrows. Representative monochromatic micrographs are shown here. C, Cells transduced with virus expressing mCherry (control) or 3xFLAG-tagged Gata4 were harvested 2 weeks later and assayed by Western blot using antibodies against alpha-smooth muscle actin (αSMA) and alpha-tubulin (α-Tubulin; loading control).</p

Effect of overexpression of <i>BAF60C</i>, <i>Gata4</i> and <i>TBX5</i> on expression of cardiac differentiation markers in human CPCs.

<p>A, Different combinations of three transcription factors (<i>BAF60C</i> [B], <i>Gata4</i> [G], and <i>TBX5</i> [T]) were introduced to c-kit+ human CPCs via lentivirus. Cells were then cultured for 2 weeks and analyzed for relative changes in mRNA levels of indicated genes using quantitative RT-PCR. Cells expressing mCherry (mCh) served as a negative control (not shown). The level of indicated transcript in each group was compared to that of mCherry-expressing control group, and is expressed as a relative fold change. For each condition, <i>n</i> = 4. Bar graphs show mean ± SEM. *, <i>p</i> < 0.05. B, Semi-quantitative RT-PCR analysis showing increased expression of the indicated transcription factors in each treatment group. β-actin served as a loading control.</p

Effect of transcription factor overexpression on the level of endogenous transcription factors.

<p>A and B, Different combinations of transcription factors (<i>Gata4</i> [G], <i>MEF2C</i> [M], <i>NKX2</i>.<i>5</i> [N], and <i>TBX5</i> [T]) were expressed in c-kit+ human CPCs via lentivirus. Cells were then cultured for 10 days and analyzed for relative changes in mRNA levels of indicated genes using quantitative RT-PCR. Cells expressing mCherry (mCh) served as a negative control (not shown). The level of indicated transcript in each group was compared to that of mCherry-expressing control group, and is expressed as a relative fold change. For each condition, <i>n</i> = 4. Bar graphs show mean ± SEM. *, <i>p</i> < 0.05. C, Representative cDNA sample from each group was analyzed by PCR for each transcription factor to demonstrate the combination of transcription factors overexpressed in each group. PCR products were then analyzed by agarose gel electrophoresis followed by ethidium bromide staining. GAPDH served as an internal control.</p

List of primers used for qPCR analysis.

<p>List of primers used for qPCR analysis.</p

Co-expression of transcription factors in combination in human CPCs.

<p>Different combinations of transcription factors (<i>Gata4</i> [G], <i>MEF2C</i> [M], <i>NKX2</i>.<i>5</i> [N], and <i>TBX5</i> [T]) were introduced to c-kit+ human CPCs via lentivirus. Cells were then cultured for 10 days and analyzed for relative changes in mRNA levels of the indicated genes using quantitative RT-PCR. Cells expressing mCherry served as a negative control (not shown). The level of indicated transcript in each group was compared to that of mCherry control group, and is expressed as a relative fold change. For each condition, <i>n</i> = 4. Bar graphs show mean ± SEM. *, <i>p</i> < 0.05.</p

Summary illustration showing the effects that <i>Gata4</i>, <i>MEF2C</i>, <i>NKX2</i>.<i>5</i> or <i>TBX5</i> overexpression have on cardiovascular differentiation potential of human CPCs.

<p>Contribution by TF overexpression to four different cardiovascular lineages (Cardiomyocyte, Endothelial, Smooth Muscle, and Mesenchymal) is shown here based on the gene expression changes induced by each TF. Large arrows indicate promoting action towards the lineage. T-shaped bar indicate inhibitory action towards the lineage. Arrows that are next to genes indicate either increase (upward) or decrease (downward) in the gene expression. Question marks indicate the questionable or inconclusive nature of the association.</p