Age distribution of human papillomavirus infection and neutralizing antibodies in healthy Chinese women aged 18-45 years enrolled in a clinical trial.

OBJECTIVES: Data from clinical trials of human papillomavirus (HPV) vaccines showed that women naïve (negative for both type-specific antibodies and DNA) to vaccine types would derive benefit from vaccination; therefore, an understanding of the proportion of naïve women in different age groups is important for developing HPV vaccination strategies. METHODS: From November 2012 to April 2013, a total of 7372 healthy women aged 18-45 years were recruited in five provinces in China. Cervical specimens and serum samples were collected for each woman at entry. Cervical specimens were first tested by the HPV DNA enzyme immunoassay method; if positive, the specimens were then tested by reverse hybridization line probe assay and HPV-16 and HPV-18 specific polymerase chain reactions. Neutralizing antibodies against HPV-16 or HPV-18 were tested with a pseudovirion-based neutralization assay. RESULTS: The overall prevalence of high-risk HPV DNA was 14.8% (1088/7367, 95% CI 14.0-15.6), and the seroprevalence of neutralizing antibodies against HPV-16 and HPV-18 was 12.6% (925/7367) and 4.9% (364/7367), respectively. In younger women (18-26 years) and middle-aged women (27-45 years), 83.8% (3116/3719) and 81.4% (2968/3648) were naïve to both HPV-16 and HPV-18 (both neutralizing antibodies and DNA were negative), respectively. In addition, 98.5% (3664/3719) and 98.0% (3575/3648) of the younger or middle-aged women were naïve to at least one HPV type (HPV-16 or HPV-18). DISCUSSION: This study revealed that the majority of Chinese women aged 18-26 years and 27-45 years were naïve to both HPV-16 and HPV-18 and would thus derive full benefit from bivalent HPV vaccination.

Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo.

Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Angiogenic factors are potentially optimal targets for therapeutic strategies because they are essential for tumor growth and progression. In this study, we sought a strategy for efficiently delivering an antisense cDNA molecule of the vascular endothelial growth factor (VEGF) to glioma cells. The recombinant adenoviral vector Ad5CMV-alphaVEGF carried the coding sequence of wild-type VEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignant glioma cells with the Ad5CMV-alphaVEGF resulted in reduction of the level of the endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein. Treatment of s.c. human glioma tumors established in nude mice with intralesional injection of Ad5CMV-alphaVEGF inhibited tumor growth. Taken together, these findings indicate that the efficient down-regulation of the VEGF produced by tumoral cells using antisense strategies has an antitumor effect in vivo. This is the first time that an adenoviral vector is used to transfer antisense VEGF sequence into glioma cells in an animal model, and our results suggest that this system may have clinical and therapeutic utility.

The relevance of cell proliferation, vascular endothelial growth factor, and basic fibroblast growth factor production to angiogenesis and tumorigenicity in human glioma cell lines.

Tumor growth is partially dependent on angiogenesis, a process that relies on angiogenic factors. Tumorigenicity of cancer cells is thought to be associated with the production of various angiogenic factors that stimulate or inhibit the rate of endothelial cell migration and proliferation. However, the relative importance of specific individual factors originally studied in cancer cell lines has yet to be determined in vivo. In this study, we examined seven human glioma cell lines for dynamic changes of two major angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and for doubling time and tumorigenicity in nude mice. Various correlation studies demonstrated that in these glioma cell lines, VEGF expression correlated well with RBC density in tumor sections (r2 = 0.804) and with average tumor weight (r2 = 0.987). In contrast, bFGF expression in the observed glioma cell lines did not correlate with tumorigenicity (r2 = 0.001) or with VEGF expression (r2 = 0.255). Furthermore, there was no correlation between doubling time and tumorigenicity in these cell lines (r2 = 0.160). Taken together, these results suggest that VEGF plays a major role in glioma formation and that down-regulation of VEGF, rather than bFGF, would be a more effective choice for glioma gene therapy.

MMAC/PTEN tumor suppressor gene regulates vascular endothelial growth factor-mediated angiogenesis in prostate cancer.

Prostate cancer presents with a broad spectrum of biologic behavior, ranging from being an indolent, incidental finding to an aggressively invasive and metastatic disease. An improved understanding of the events involved in prostate cancer progression is critically important to its diagnosis and staging, as well as to the development of new therapies. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of an angiogenic, gene-driven switch. In prostate cancer, one of the most powerful stimulators of angiogenesis is the vascular endothelial growth factor (VEGF). VEGF transcription can be induced by hypoxia through activation of the PI3 kinase pathway and hypoxia-inducible factor alpha. MMAC/PTEN (henceforth referred to as PTEN) is a recently identified tumor suppressor gene residing on chromosome 10q23, which is frequently inactivated in a wide range of human tumors, including advanced prostate cancer. The goal of this study was to determine whether PTEN inhibits angiogenesis by modulating VEGF activity. Our results showed that reintroduction of the PTEN gene into human prostate PC-3 and LNCaP cells decreased VEGF secretion, which was accompanied by various biologic activities, including inhibited endothelial cell growth and migration. PTEN expression also down-regulated VEGF mRNA levels, as detected by RT-PCR analysis. Concomitant with lessened VEGF expression was the reduction of VEGF promoter activity in PTEN-expressing cells. Our findings suggest that PTEN modulates angiogenesis by regulating VEGF expression.

A novel approach to glioma gene therapy: down-regulation of the vascular endothelial growth factor in glioma cells using ribozymes.

Glioblastoma multiforme is one of the most highly vascularized solid neoplasms, therefore treatments that target neovascularization process would be of great clinical importance. Studies of glioblastoma angiogenesis have revealed that expression of the vascular endothelial growth factor (VEGF) is up-regulated in these tumors. Previous reports have shown that down-regulation of VEGF correlates with modification in the glioma growth. To examine this phenomenon further, in this study we constructed two hammerhead ribozymes (RZI and RZII) to target the 5' common region of VEGF mRNA. Both ribozymes exhibited site-specific cleavage to a 318-nucleotide VEGF transcript and showed a high digestion efficiency in vitro (65-95%). After the transfection of glioma cells with two expression vectors carrying the ribozyme sequence, Northern blot analyses detected high levels of ribozyme expression. Treatment of the glioma cells with the ribozymes resulted in a reduction in VEGF mRNA in six of eight clones. Furthermore, the anti-VEGF effect was confirmed at protein level. Thus, enzyme-linked immunoabsorbent analyses (ELISA) showed a >70% reduction in the VEGF165 expression level. These results indicate that hammerhead ribozymes may be useful in down-regulating VEGF expression and suggest that anti-VEGF strategies may be used to potentiate other gene therapies targeting tumor suppressor genes.

Expression of human papillomavirus E7 mRNA in human oral and cervical neoplasia and cell lines.

Human papillomaviruses (HPVs) have been strongly linked to progression of human cancers, such as cervical and oral cancers. Two HPV oncoproteins, E6 and E7, can inhibit the tumor suppressor proteins, p53 and pRB, respectively, resulting in a deregulation of the cell cycle. In order to further test the significance of HPV expression in oral and cervical carcinogenesis, we analyzed HPV E7 mRNA in oral and cervical neoplasia and cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). We found that HPV E7 mRNA was present in 90% of patients with oral neoplasia and 100% of patients with cervical neoplasia. Quantitative RT-PCR and western blot analysis on both transformed cervical and oral epithelial cell lines demonstrated that the mRNA level of HPV-16 E7 corresponded to E7 protein level, suggesting that HPV oncogene expression is primarily regulated at the transcriptional or post-transcription level. The potential clinical application of quantitative RT-PCR for HPV E7 mRNA expression in cancer screening and treatment evaluation requires further investigation.

Correlation of cisplatin sensitivity with differential alteration of EGFR expression in head and neck cancer cells.

Overexpression of human epidermal growth factor (EGFR) has been associated with a variety of human malignancies. The exact role of EGFR in human malignancies and its correlation with chemotherapeutiveness response has not been determined. Using a quantitative RT-PCR method, we previously studied the effects of cisplatin treatment on levels of EGFR mRNA in human papillomavirus (HPV)-positive head and neck cancer cell lines. In this report we extended these studies to HPV-negative head and neck cancer cells. We also compared the growth inhibition and 50% inhibitory concentration (IC50) of cisplatin between these cells. We found that three of four HPV-negative cell lines had 3 to 5 times higher cisplatin IC50 values as compared to two HPV-positive cell lines. EGFR mRNA levels were increased after exposure to cisplatin in the cell lines with the higher IC50 values, while EGFR levels were reduced after cisplatin exposure in the cell lines with the lower IC50 values. These results suggest that the cisplatin sensitivity of head and neck cancer cells corresponds to subsequent alteration of EGFR levels following cisplatin treatment.

Sequence evolution in mitochondrial ribosomal and ND-1 genes in lepidoptera: implications for phylogenetic analyses.

A 2,256-bp sequence of the mitochondrial genome of a lepidopteran (Spodoptera frugiperda) contains tRNAs for valine and leucine, the 16S rRNA, and three-quarters of the ND-1 presumptive protein-coding gene. A 64-bp stretch of unknown function was located between the rRNA and leucine tRNA. Sequence divergence in the 16S rRNA obtained from alignment with published insect sequences is consistent with phylogenetic hypotheses, in that Diptera and Lepidoptera are more closely related to each other (24% sequence divergence) than either is to Hymenoptera (31%). Within the ND-1 gene, sequences for four additional Lepidoptera were generated for a 314-bp region and contrasted with published sequences for the locust and Drosophila. Sequence divergence in this region was consistent with accepted phylogenetic relationships, but results of parsimony analyses were not. Cladograms consistently recovered accepted higher level relationships (monophyly of Lepidoptera), despite high homoplasy, but were unable to resolve superfamily and family relationships within Lepidoptera, regardless of the outgroup or character subset analyzed. Character analysis indicated that homoplasy was decreased at higher levels when first- and second-codon sites were used exclusively. At the lowest level (families), resolution was enhanced by inclusion of third-codon sites. Inability of molecular data to recover a well-established phylogeny may be rectified by additional characters or taxa, but it is clear that homoplasy is sufficiently high to caution against the acceptance of relationships generated with this molecular region that are not extremely robust.

A rapid, two-step method for high-yield purification of recombinant rat acidic and basic fibroblast growth factors.

We describe the preparation of vectors for T7 polymerase-driven, high-level expression of rat acidic (aFGF) or basic (bFGF) fibroblast growth factors in Escherichia coli. Following isopropyl-beta-D-thiogalactoside induction of T7 polymerase, rat aFGF or bFGF represented a major portion of the proteins synthesized by vector-transformed E. coli. Passage of cell extracts through an Amicon YM-100 membrane provided ultrafiltrates containing either aFGF or bFGF as the principal component. A single heparin-Sepharose chromatography of the ultrafiltrates yielded essentially homogenous, biologically active, recombinant rat aFGF or bFGF. By silanizing vessels and using buffers containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, we could store homogenous aFGF or bFGF preparations without significant loss during repeated freezing and thawing. Previous methods for purifying aFGF or bFGF utilized salt fractionation followed by sequential ion exchange and heparin-Sepharose chromatography. These purified aFGF or bFGF preparations routinely were stored in buffered carrier protein to minimize mitogen loss resulting from adsorption to glass or plastic surfaces. In contrast, the methods that we detail are rapid, require minimal manipulation of preparations, and permit storage of carrier-free, homogenous preparations without loss resulting from surface adsorption. The protocols can be used for preparation of homogenous aFGF or bFGF of other species and may be readily applied to the isolation and characterization of FGF-like mitogens present in cultured cell extracts, conditioned medium, or tissue preparations.

A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards.

The quantitative measurement of gene expression requires consistent and reliable standards. At least two categories of standards, endogenous and exogenous, are currently used for quantitative PCR. The reliability of these two methods, however, has not been carefully compared. We hypothesized that a reliable quantitative PCR assay would be able to detect known dilutions of a given single-stranded (ss-) cDNA. By measuring VEGF ss-cDNA copy numbers or signal ratios of GAPDH/VEGF in 10x and 100x diluted samples of two original ss-cDNA preparations, an exogenous recombinant DNA standard (a VEGF-mimic plasmid) and an endogenously expressed GAPDH standard were tested for their ability to detect dilution factors. Using the recombinant DNA standard, the dilution factor was detected as 10.3 and 135.0 in 10x and 100x diluted samples of the original CaSki cell ss-cDNA, respectively. The detected dilution factors were 12.3 and 226.2, respectively, in 10x and 100x diluted ss-cDNA from U-251 MG cells. On the other hand, with the endogenous GAPDH standard, the dilution factors were detected as 2.7 and 8.0 in the same 10x and 100x dilutions of the original U-251 MG cell ss-cDNA. Using the same endogenous GAPDH standard, the detected dilution factors were both 4.8 in 10x and 100x dilutions of the original CaSki cell ss-cDNA. It was also found that the number of endogenous copies of GAPDH mRNA was about 1000 times higher than VEGF. The high internal lockup ratio of GAPDH vs VEGF copy numbers and the requirement for additional primer pairs make the use of an abundant endogenous standard an unreliable choice in quantitative or semi-quantitative PCR. In contrast, exogenous standard-based quantitative PCR was shown to be an accurate and reliable method for the quantitation of gene expression.

Rat prostate cancer cell line-specific production and apparent secretion of heparin-binding growth factors.

Cultured C3 and T5 AXC/SSh rat prostate cancer cells and their conditioned media contained 1000-fold more heparin-binding mitogens than did normal AXC/SSh rat prostates. Immunological analyses confirmed that rat ventral prostate contained only a 17.5 kilodalton (kDa) basic fibroblast growth factor (bFGF)-like mitogen. In contrast, combined immunological and metabolic radiolabeling analyses showed that C3 cells contained 23/24 and 14 kDa bFGF-like polypeptides, whereas the principal bFGF-like polypeptides of C3 cell conditioned medium were proteins having masses of 17.5 and 14 kDa. Identical analyses showed that T5 cells contained 17.5 and 14 kDa bFGF-like polypeptides, whereas the principal bFGF-like polypeptides of T5 cell conditioned medium were proteins having masses of 17.5, 16, and 14 kDa. We found that bFGF-like proteins of mass less than 17.5 kDa, which were present in conditioned medium, were not derived by proteolysis of higher molecular weight bFGF-like mitogens after these were processed into conditioned medium. Northern analyses showed that normal prostate and prostate cancer cells contained acidic fibroblast growth factor transcripts of 6.5 and 3.4 kilobases (kb) and bFGF transcripts of 6.0 and 2.5 kb. Prostate cancer cells also contained a 12-kb bFGF transcript that was not present in normal prostate. Southern analysis of restriction endonuclease-digested normal prostate or prostate cancer cell genomic DNA showed that the 12-kb bFGF transcript was not the product of a rearranged bFGF gene. Our data show that rat prostate cancer cells contain bFGF-like polypeptides of mass 14 to 23/24 kDa and suggest that these cells secrete bFGF-like polypeptides.

Differential expression of epidermal growth factor receptor in human head and neck cancers.

BACKGROUND: Over-expression of human epidermal growth factor receptor (EGFR) is associated with a variety of human malignancies, including head and neck cancer. It has also been studied for its effect on cancer cell responses to chemotherapy. To accurately measure changes in EGFR expression that might be of diagnostic or prognostic importance in head and neck cancers, a quantitative assay for the direct detection of EGFR messenger ribonucleic acid (mRNA) was developed. METHODS: Our method was based on competitive reverse transcriptase-polymerase chain reaction (RT-PCR) that was able to measure EGFR mRNA levels undetectable by northern-blot analysis. We measured EGFR mRNA by RT-PCR in human head and neck cancers and their corresponding adjacent, histologically normal tissues and in cisplatin-treated and untreated oral epithelial cell lines. RESULTS: All the tumor samples had higher EGFR mRNA levels than their corresponding adjacent normal tissues. It is also shown that EGFR mRNA levels in normal oral epithelial cells were elevated after exposure to cisplatin. In contrast, EGFR mRNA levels in oral cancer cells were decreased after the exposure, suggesting that increased EGFR expression may have different functions in cancer cells and in normal cells under stress. CONCLUSIONS: Accurate monitoring of EGFR expression may be a useful marker for diagnosis, treatment, and prognosis of head and neck cancer.

Endogenous fibroblast growth factor-1 or fibroblast growth factor-2 modulate prostate cancer cell proliferation.

We constructed expression vectors containing either rat fibroblast growth factor (FGF)-1 for FGF-2 cDNA cloned in either the sense orientation or antisense orientation relative to the metallothionein promoter of plasmid pMTneo.1. Stable AXC/SSh rat prostate cancer cell transfectants expressing either chimeric FGF-1-sense, chimeric FGF-1-antisense, or chimeric FGF-2-antisense transcripts were obtained. Stable transfectants expressing chimeric FGF-2-sense transcripts were not obtained. Control, sense, and antisense transfectants expressed endogenous FGF-1 and endogenous FGF-2 transcripts, implying that transfection did not eliminate endogenous FGF transcripts. Control transfectants and sense transfectants contained FGF-1 isoforms having a mass of 16.4 or 17.3 kDa and FGF-2 isoforms having a mass of 17, 19.5, or 21.5 kDa. Significantly, adult AXC/SSh rat prostate contained only the 17.3 kDa FGF-1 isoform and the 17 kDa FGF-2 isoform, indicating that neoplastic transformation was associated with elaboration of novel, prostate epithelial cell-derived FGF-2 isoforms. FGF-1 antisense RNA expression eliminated transfectant FGF-1 isoforms without affecting FGF-2 isoform content. Similarly, FGF-2-antisense RNA expression eliminated the transfectant 21.5 kDa FGF-2 isoform, diminished the 19.5 kDa FGF-2 isoform content, and reduced the 17 kDa FGF-2 isoform content to barely detectable levels without affecting the FGF-1 isoform content. This established that FGF-antisense RNAs specifically inhibited translation of cognate, endogenous FGF transcripts. Doubling times of control transfectants and sense transfectants were indistinguishable and were not affected by including FGF-1 or FGF-2 in the culture medium. Doubling times of FGF-1-antisense or FGF-2-antisense transfectants were 1.3- to 1.4-fold greater than those of control transfectants or sense transfectants, and either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling times to values indistinguishable from those of control transfectants or sense transfectants. This established that with regard to prostate cancer cell proliferation: (a) endogenous FGF-1 cannot substitute for endogenous FGF-2 eliminated by FGF-2-antisense RNA expression; and (b) endogenous FGF-2 cannot substitute for endogenous FGF-1 eliminated by FGF-1-antisense RNA expression. In contrast, either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling time. The results of these studies establish that endogenous FGF-1 and endogenous FGF-2 modulate prostate cancer cell proliferation and imply that FGF-1 and FGF-2 of endogenous and exogenous origin conjointly control aspects of prostate cancer cell homeostasis. Our findings suggest complex interaction between components of prostate cancer cell regulatory processes and endogenously produced and exogenously accessible FGF-1 and FGF-2.