Cigarette smoking and cervical intraepithelial neoplasia among colposcopy patients

Epidemiologic and biochemical evidence suggest that smoking is an independent risk factor for cervical neoplasia; however, only two studies have adjusted by the potential confounding effect of human papillomavirus (HPV). To determine the association between self-reported current cigarette smoking and cervical intraepithelial neoplasia (CIN), we conducted a case-control study that controlled for HPV infection and other reported risk factors. The medical records of all new patients referred to the University of Texas M. D. Anderson Cancer Center (UTMDACC) Colposcopy Clinic were reviewed. The study population (n = 564) consisted of all white, black, and Hispanic non-pregnant women who were residents of Texas, and had no history of treatment for cervical neoplasia. Cases (n = 313) included women diagnosed at the UTMDACC with CIN; while controls (n = 251) included those patients diagnosed at the colposcopy clinic as non-CIN (negative 47%, inflammation or atypia 25%, and koilocytosis 27%). Diagnosis was based on a colposcopically directed biopsy in 95% of the subjects, and all subjects were tested for HPV by dot blot hybridization. The crude odds ratio for cigarette smoking and CIN was 1.37 (95% CI 0.97-1.95); however, after adjusting for HPV, age, education, race, number of sexual partners, and age at first sexual intercourse, the odds ratio decreased to 0.91 (95% CI 0.61-1.41). A higher crude odds ratio was observed with CIN 3 (OR = 1.75, 95% CI 1.08-2.83), but this effect also disappeared after adjustment (OR = 1.06, 95% CI 0.57-1.96). Similar results were observed when controlling only for HPV: OR = 1.11 (95% CI 0.77-1.59) for CIN combined and 1.25 (95% CI 0.76-2.08) for CIN 3. These findings suggest that cigarette smoking is not an independent risk factor for CIN in this population, and that HPV may be an important confounding factor for this association. ^

Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

The performance of human papillomavirus high-risk DNA testing in the screening and diagnostic settings.

OBJECTIVE: We sought to evaluate the performance of the human papillomavirus high-risk DNA test in patients 30 years and older. MATERIALS AND METHODS: Screening (n=835) and diagnosis (n=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II (HCII) test result with the worst histologic report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR-), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear. RESULTS: For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR- were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR- were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing. CONCLUSIONS: The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older.

Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions.

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.

Molecular mechanisms of prostatic carcinoma growth and progression

Prostate cancer represents the most commonly diagnosed malignancies in American men and is the second leading cause of male cancer deaths. The overall objectives of this research were designed to understand the cellular and molecular mechanisms of prostatic carcinoma growth and progression. This dissertation was divided into two major parts: (1) to clone and characterize soluble factor(s) associated with bone that may mediate prostatic carcinoma growth and progression; (2) to investigate the roles of extracellular matrix in prostatic carcinogenesis.^ The propensity of prostate cancer cells to metastasize to the axial skeleton and the subsequent osteoblastic reactions observed in the bone indicate the possible reciprocal cellular interaction between prostate cancer cells and the bone microenvironment. To understand the molecular and cellular basis of this interaction, I focused on the identification and cloning of soluble factor(s) from bone stromal cells that may exert direct mitogenic action on cultured prostate cells. A novel BPGF-1 gene expressed specifically by bone and male accessory sex organs (prostate, seminal vesicles, and coagulating gland) was identified and cloned.^ The BPGF-1 was identified and cloned from a cDNA expression library prepared from a human bone stromal cell line, MS. The conditioned medium (CM) of this cell line contains mitogenic materials that induce human prostate cancer cell growth both in vivo and in vitro. The cDNA expression library was screened by an antibody prepared against the mitogenic fraction of the CM.^ The cloned BPGF-1 cDNA comprises 3171 nucleotides with a single open reading frame of 1620 nucleotides encoding 540 amino acids. The BPGF-1 gene encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity in human cells and tissues, but only one transcript (2.5 kb) in rat and mouse tissues. Southern blot analysis of human genomic DNA revealed a single BPGF-1 gene. The BPGF-1 gene is expressed predominantly in bone and seminal vesicles, but at a substantially lower level in prostate. Polyclonal antibodies generated from synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a putative BPGF-1 protein with an apparent molecular weight of 70 kDa. The conditioned media isolated from COS cells transfected with BPGF-1 cDNA stimulated the proliferation and increased the anchorage-independent growth of prostate epithelial cells. These findings led us to hypothesize that BPGF-1 expression in relevant organs, such as prostate, seminal vesicles, and bone, may lead to local prostate cancer growth, metastasis to the seminal vesicles, and subsequently dissemination to the skeleton.^ To assess the importance of extracellular matrix in prostatic carcinogenesis, the role of extracellular matrix in induction of rat prostatic carcinoma growth in vivo was evaluated. NbE-1, a nontumorigenic rat prostatic epithelial cell line, was induced to form carcinoma in athymic nude hosts by coinjecting them with Matrigel and selected extracellular matrix components. Induction of prostatic tumor formation by laminin and collagen IV was inhibited by their respective antibodies. Prostatic epithelial cells cloned from the tumor tissues were found to form tumors in athymic nude hosts in the absence of exogenously added extracellular matrix. These results suggest that extracellular matrix induce irreversibly prostatic epithelial cells that behave distinctively different from the parental prostatic epithelial cell line. ^

Molecular markers of human papillomavirus (HPV) infection and epidemiologic risk factors for squamous intraepithelial lesions (SIL) of the cervix

Infection with certain types of HPV is a necessary event in the development of cervical carcinoma; however, not all women who become infected will progress. While much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation, little is known about the additional factors needed to drive the process. Currently, conventional cervical screening is insufficient at identifying women who are likely to progress from premalignant lesions to carcinoma. Aneuploidy and chromatin texture from image cytometry have been suggested as quantitative measures of nuclear damage in premalignant lesions and cancer, and traditional epidemiologic studies have identified potential factors to aid in the discrimination of those lesions likely to progress. ^ In the current study, real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, LSIL, and HSIL. Quantitative cytometry was used to gather information about the DNA index and chromatin features of cells from the same women. Logistic regression modeling was used to establish predictor variables for histologic grade based on the traditional epidemiologic risk factors and molecular markers. ^ Prevalence of mRNA transcripts was lower among women with normal histology (27%) than for women with LSIL (40%) and HSIL (37%) with mean levels ranging from 2.0 to 4.2. The transcriptional activity of HPV 18 was higher than that of HPV 16 and increased with increasing level of dysplasia, reinforcing the more aggressive nature of HPV 18. DNA index and mRNA level increased with increasing histological grade. Chromatin score was not correlated with histology but was higher for HPV 18 samples and those with both HPV 18 and HPV 16. However, chromatin score and DNA index were not correlated with mRNA levels. The most predictive variables in the regression modeling were mRNA level, DNA index, parity, and age, and the ROC curves for LSIL and HSIL indicated excellent discrimination. ^ Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from cervical swabs. Epidemiological modeling of malignant progression in the cervix should include molecular markers, as well as the traditional epidemiological risk factors. ^

Transformation studies of a nontumorigenic rat prostatic epithelial cell line with activated {\it myc\/} and {\it neu\/} oncogenes

A cloned nontumorigenic prostatic epithelial cell line, NbE-1.4, isolated from Noble (nbl/crx) rat ventral prostate, was used to examine the potential role of activated myc and neu oncogenes in prostate carcinogenesis. Transfection of SV40 promoter/enhancer driven constructs containing either v-myc, truncated c-myc, or neu-T (activated neu) oncogenes was accomplished using calcium phosphate-mediated DNA transfer. Cells were cotransfected, as necessary, with pSV2neo, allowing for selection of positive clones using the antibiotic geneticin (G418). G418 resistant colonies were pooled in some cases or limiting dilution exclusion cloned in others as described. Transfection of NbE-1.4 cells with activated myc oncogenes resulted only in the partial transformation. These cells display an altered morphology and decreased dependence on serum factors in vitro; however, saturation density, soft agar colony formation and growth assay in male athymic nude mice were all negative. Transfection and overexpression of NbE-1.4 cells with an activated neu oncogene alone resulted in tumorigenic conversion. Cell transformation was evident following an examination of the altered cellular morphology, an increased soft agar colony formation, and an acquisition of a tumorigenic potential when injected s.c. into male athymic nude mice. neu-transformed NbE-1.4 cells displayed elevated activity of the neu receptor tyrosine kinase. Furthermore, qualitative changes in tyrosine phosphorylated proteins were found in neu transformed cell clones. These changes were associated with elevated expression of mRNAs for laminin $\beta$1, $\beta$2, and procollagen type IV. The expression of fibronectin and E-cadherin, which are often lost during tumorigenesis, did not correlate with the tumorigenic phenotype. Therefore, it appears that neu oncogene overexpression has been found to be associated with the transformation of rat prostatic epithelial cells, presumably through alterations in gene expression that regulate extracellular matrix. The possible interrelationship and functional significance between neu oncogene expression and the elevated extracellular matrix gene expression is discussed. ^