Racial/ethnic differences in HPV 16/18 genotypes and integration status among women with a history of cytological abnormalities.

OBJECTIVE: HPV genotype distribution varies by race/ethnicity, but is unclear whether there are racial/ethnic variations in HPV 16/18 integration in the host genome. We describe HPV16/18 infection and integration status in a racially/ethnically diverse sample of women with a recent abnormal Pap test. METHODS: Patients (n=640) represent a subset of women participating in a clinical trial. Cervical swabs were tested for HPV16/18 DNA using type-specific polymerase chain reaction assays. Viral integration status was assessed using type-specific integration assays and categorized as fully integrated, fully non-integrated, or mixed. Unconditional logistic regression was used to generate unadjusted (OR) and adjusted odds ratios (aOR) to assess the association between self-reported race/ethnicity and risk of these outcomes. RESULTS: Hispanic and non-Hispanic black women had half the odds of prevalent HPV16 compared to non-Hispanic white women (aORs: 0.43 and 0.45, respectively). The prevalence odds of HPV18 was less than half among Hispanic women (aOR: 0.48), but not significantly different between black and white women (aOR: 0.72). Among women with prevalent HPV16, the odds of fully integrated viral DNA were significantly higher among black women (aORs: 2.78) and marginally higher among Hispanic women (aOR: 1.93). No racial/ethnic differences were observed for HPV18 DNA integration. CONCLUSIONS: While HPV16 and 18 infections were less prevalent among Hispanic and black women compared to whites, their HPV16 DNA was more likely to be present in a fully integrated state. This could potentially contribute to the higher rates of abnormal cytology and cervical dysplasia observed among Hispanic and black women.

Immune response to hepatitis B surface antigen: enhancement by prior injection of antibodies to the idiotype.

Anti-idiotype reagents that recognize a common idiotype associated with the combining site of antibodies to hepatitis B surface antigen (anti-HBs) were used to manipulate the immune response to hepatitis B surface antigen in BALB/c mice. The injection of antibodies to the idiotype before antigenic stimulation resulted in an increase in the number of cells secreting immunoglobulin M antibodies to hepatitis B surface antigen. Anti-HBs-secreting cells were also induced by administration of antibodies to the idiotype without subsequent antigen exposure. These findings indicate that the immune response to hepatitis B surface antigen in mice is regulated through an idiotype-anti-idiotype network.

Effect on cancer cells of plasmids that express antisense RNA of human papillomavirus type 18.

Some human squamous cell carcinomas contain DNA of human papillomaviruses (HPV) and express RNA from the E6 and E7 genes. We have examined the effect of plasmids that express antisense RNA of these genes on the growth of the human cancer cell lines HeLa, C4-1, and 1483, which contain HPV type 18 DNA. As controls, the human cancer cell line 183 and the Vero line of monkey kidney cells were used, which do not contain HPV. Plasmids were introduced into the cells by electroporation; cells that contained HPV type 18 accepted the antisense-expressing plasmids at a lower frequency than the cells that lacked HPV. Cell lines were developed from HeLa cells that contained sense- or antisense-expressing plasmids, and lines that contained antisense-expressing plasmids showed slower growth, reduced ability to form colonies in soft agar, and increased serum requirements. The use of antisense HPV RNA might be a suitable approach to gene therapy of HPV-expressing human cancers.

Interleukin-1 regulates interleukin-6 secretion in human oral squamous cell carcinoma in vitro: possible influence of p53 but not human papillomavirus E6/E7.

We have previously shown that interleukin-1 (IL-1) and IL-6 are constitutively produced by human oral squamous cell carcinoma (SCC) and some derived cell lines but not by cultured normal oral keratinocytes. To elucidate possible cytokine regulatory pathways that may contribute to oral SCC growth and/or progression, we tested the hypotheses that exogenous and/or endogenous IL-1 regulates IL-6 production in vitro. We investigated the effects of exogenous IL-1 and IL-6 on secondary cytokine secretion. Our studies revealed that IL-1 strongly up-regulated IL-6 protein secretion in all three cell lines tested. This effect was completely abrogated by IL-1 receptor antagonist. IL-1 receptor antagonist also inhibited the secretion of IL-1alpha and IL-1beta in two of three cell lines. These data show for the first time that IL-1 strongly up-regulates IL-6 and support the notion of autocrine regulation of IL-1 in certain oral SCC cell lines. Additionally, because human papillomavirus (HPV) infection and p53 mutation have been implicated in the malignant transformation of SCC, we explored a second hypothesis, that HPV and/or p53 mutation contribute to cytokine dis-regulation. We investigated HPV DNA presence, transcriptional activation of HPV E6/E7 (in HPV DNA-positive cell lines), and p53 gene status in our cell lines. No association between HPV DNA and cytokine expression was found. However, the oral SCC cell lines secreting the most IL-6 had mutant rather than wild-type p53.

Head and neck squamous cell growth suppression using adenovirus-p53-FLAG: a potential marker for gene therapy trials.

The recombinant wild-type p53 adenovirus has been proven effective against the growth of human head and neck squamous cell cancer (SCCHN) cell lines iir vitro and in a nude mouse model. The addition of a FLAG peptide sequence was used in this study, along with the p53 adenovirus vector as a marker of the site of the gene therapy activity. It provides clear evidence of the exogenous gene product within the transduced carcinoma cells. No alterations in transcription or translation of the p53 gene product were noted with the addition of the FLAG sequence to the original p53 adenovirus vector. Immunohistochemical analysis displayed simultaneous expression of the p53 and FLAG proteins in the infected cells. The p53 protein remained localized to the nucleus, whereas the FLAG protein was additionally noted in the cytoplasm. In vitro growth suppression assays and in vivo microscopic residual tumor model experiments in nude mice showed a similar tumoricidal effect with the p53-FLAG adenovirus vector to that with the previously studied p53 adenovirus vector without the addition of the FLAG sequence. We conclude that the addition of the FLAG octapeptide sequence allows identification of those cells that have been affected by the molecular therapy independent of the endogenous gene expression of the cells. This novel molecular tracer may prove useful in characterizing infection efficiency and in gene therapy trials.

Effects on tumor cells of ribozymes that cleave the RNA transcripts of human papillomavirus type 18.

Many human cervical and oral carcinomas express RNA of human papillomaviruses, and the RNA transcript provides a potential target for gene therapy of these carcinomas. Three hammerhead ribozymes that were targeted to RNA of human papillomavirus type 18 (HPV-18) were cloned into a plasmid expression vector. Each plasmid was then transfected into the HPV-18-expressing cell line. HeLa, or the non-HPV-expressing oral cancer cell line, Tu167. None of the ribozymes had any effect on the phenotype of Tu167 cells. In contrast, each ribozyme affected the phenotype of HeLa cells, causing reduced growth rates, increased serum dependency, and reduced focus formation in soft agar. A molecule that had the same antisense sequences as a ribozyme but lacked the catalytic sequences affected the HeLa cell phenotype to a much lesser extent. The effects of two of the ribozymes could be attributed in part to an increased intracellular concentration of the tumor suppressor protein p53. The most effective ribozyme was targeted to nucleotide 309 in the HPV-18 transcript, but each of the three ribozymes appears to have potential for gene therapy of cancers that express HPV-18.

Endogenous p53 gene status predicts the response of human squamous cell carcinomas to wild-type p53.

Prior reports suggest that p53 protein status may influence the response to gene transduction with wild-type (wt) p53. Adenoviral vectors containing the p53 gene were administered to normal keratinocytes, to squamous cell carcinoma (SCC) lines with varied p53 protein status (absent, mutant, wt, or degraded by papillomavirus), as well as to tumors formed in severe combined immunodeficient mice. The percentage of cells undergoing apoptosis, G1 growth arrest, WAF1/p21 induction, and in vivo tumor progression were studied after wt p53 gene transduction. Apoptosis developed first in normal keratinocytes, next in SCCs lacking p53 protein, and last in SCCs with mutant or degraded p53 protein. All of the cell lines studied demonstrated an increase in WAF1/p21 protein, but only those lacking p53 protein showed G1 arrest. Tumors lacking p53 protein were more susceptible to p53 overexpression than those containing mutant or degraded p53 protein. The endogenous p53 protein status of SCCs appears to influence the outcome of p53 gene transduction.

Complement potentiation of phagocyte-mediated ADCC of HSV-infected corneal cells.

The authors have shown previously that rabbit alveolar macrophages, peritoneal exudate macrophages, and polymorphonuclear leukocytes are highly effective mediators of antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected rabbit stromal keratocytes. Using a completely homologous system of nonimmune rabbit phagocytes, anti-HSV serum, complement, and HSV-infected stromal keratocyte targets, the authors have found significant potentiation of ADCC by complement. Lymphocyte-mediated ADCC, on the other hand, was not potentiated by complement. Using a 1:100 dilution of complement (noncytolytic in antibody-mediated lysis) and a 1:10 dilution of antiserum obtained from rabbits undergoing stromal keratitis, ADCC mediated by the three phagocytic cell types was augmented by approximately 22%. For example, using alveolar macrophages as effector cells, the addition of complement increased the level of ADCC From 40% to 60%, a 20% increase. Percent ADCC was augmented less by complement at higher antiserum dilutions. However, the percent of the total ADCC attributable to complement potentiation increased at these higher antiserum dilutions. In some cases, ADCC was not detectable at these high antiserum dilutions without the added complement. The potentiating effect was observed at complement dilutions as high as 1:400. Further dilution or heat-inactivation resulted in ADCC values comparable to those obtained without complement. Target cells exposed to complement alone were not killed by phagocytes.

Immune destruction of rabbit corneal cells infected with herpes simplex virus: lymphocyte reactivity by antibody-dependent cellular cytotoxicity.

Continuous rabbit cornea cells and cells grown from the stromal layer of corneas excised from New Zealand white rabbits were infected with herpes simplex virus (HSV) strain RE and examined for cytolysis (51Cr release) by antibody-dependent cellular cytotoxicity (ADCC). These sources of lymphocytes from normal animals were used as effector cells: blood, spleen, and lymph nodes. Specific activity was observed in over 75% of rabbits with all three effector cell types, using immune serum from a rabbit a 21 days after infection. Activity was generally weak (less than 30% specific 51Cr release) and was abolished by dilutions of antibody 1:1000 or greater. Effector cells were not active when nonimmune serum was substituted for immune serum. Peritoneal exudate macrophages used as controls exhibited higher levels of release and were active with antibody diluted greater than 1:1000. HSV antibody reactive in ADCC appeared 7 days after intrastromal injection of infectious virus. Lymphocyte ADCC activity obtained from draining lymph nodes of infected animals was similar to that obtained with normal animals. Results indicate a weakly active lymphocyte effector-cell system in normal and infected rabbits.

Tumor differentiation-dependent local immunity in human head and neck cancers.

Distribution of markers of local cell-mediated immunity was examined in oral tumors exhibiting different histological stages of differentiation. Using a RT-PCR-based semiquantitative technique we determined levels of Langerhans cells, CD4- and CD8-positive T-cells, macrophages/NK cells, beta2-microglobulin and IFN-gamma mRNAs from tissue biopsies. A positive correlation was found between levels of these immunological markers and the tumor differentiation stage. Since tumor differentiation may correlate with the prognosis and response to various treatment modalities, our results may be useful clinically.

Zinc-alpha2-glycoprotein expression as a marker of differentiation in human oral tumors.

Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.

Human papillomavirus associated with vaginal intraepithelial neoplasia in women exposed to diethylstilbestrol in utero.

Five out of 959 young women, exposed to diethylstilbestrol (DES) in utero, developed vaginal intraepithelial neoplasia while they were under follow-up in the Diethylstilbestrol-Adenosis Project at Baylor College of Medicine, Houston, Texas. We suggest that the development of the vaginal intraepithelial neoplasia at a younger age than usual may be caused by a higher susceptibility of the DES-exposed patient to factors associated with the development of intraepithelial neoplasia. A common finding in all five women was the detection of the deoxyribonucleic acid (DNA) of human papillomavirus types 6 or 16 in their lesions, using high-stringency in situ hybridization. The role of human papillomavirus and herpes simplex virus in the etiology of intraepithelial neoplasia is discussed. Close follow-up is recommended for DES-exposed patients, especially those who have risk factors known to be associated with genital neoplasia.

Detection of human papillomavirus DNA before and after development of invasive vulvar cancer.

Biopsy and surgical specimens were studied from two patients with squamous cell carcinoma in situ of the vulva and one with verrucous carcinoma of the vulva. All three patients subsequently developed invasive squamous cell carcinoma of the vulva. In two, human papillomaviruses (HPVs) 16 and 18 were found in the lesions before the development of invasive carcinoma. In the specimens with invasive squamous cell carcinoma, HPV 16 or 18 or both was found. The third patient had HPV 18 DNA in the specimen demonstrating squamous cell carcinoma in situ; when invasive squamous cell carcinoma was diagnosed 9 years later, HPVs 16 and 18 were found in the latter lesion. These findings lend support to the role of HPVs 16 and 18 in the development of invasive squamous cell carcinoma of the vulva.

Human papillomavirus DNA in tissue biopsy specimens of vulvar vestibulitis patients treated with interferon.

Thirteen women with the diagnosis of vulvar vestibulitis based on clinical symptoms, presence of vestibular tenderness on physical examination, and acetowhite changes of the vulvar vestibule were treated with intradermal injection of alpha-interferon. Biopsies of the acetowhite areas were analyzed for human papillomavirus (HPV) DNA using polymerase chain reaction amplification and dot blot hybridization. Eleven of 13 subjects harbored one of the HPV DNA types; six of these were type 16 and/or 18 and the others were unidentified. Five subjects (all HPV DNA-positive) reported resolution of symptoms with interferon therapy. Our results indicate the presence of HPV DNA in a subset of patients with vulvar vestibulitis, but its presence is not predictive of response to interferon therapy.

A micro solid-phase radioimmunoassay for detection of herpesvirus type-specific antibody: parameters involved in standardization.

A micro solid-phase radioimmunoassay (micro-SPRIA) was developed to demonstrate type-specific antibodies to herpes simplex virus types 1 and 2 (HSV1 and HSV2). Glycoproteins from the 123,000 dalton region of HSV1 (VP123) and the 119,000 dalton region of HSV2 (VP119) were isolated on preparative polyacrylamide gels for use as antigens in the micro-SPRIA. Human sera selected from clinical samples by virological history and appropriate microneutralization data were used to standardize the micro-SPRIA. Optimization of the assay required the use of siliconized microtiter wells for adsorption of antigen. Maximized results were highly dependent on the concentrations of antigen, primary antibody, and secondary antibody as well as the diluents used for these principal test reagents. Incorporation of HSV glycoproteins of each respective type with the optimal condition established in this study facilitates the direct detection of type-specific antibody in human sera.

A micro solid-phase radioimmunoassay for detection of herpesvirus type-specific antibody: specificity and sensitivity.

The specificity and sensitivity of a micro solid-phase radioimmunoassay (micro-SPRIA) that detects type-specific IgG antibody to herpes simplex virus types 1 and 2 (HSV1 and HSV2) were evaluated. Glycoproteins VP123 (molecular weight, 123,000) of HSV1 and VP119 (molecular weight, 119,000) of HSV2 were found to display the greatest degree of antigenic type-specificity of several HSV antigens tested with the micro-SPRIA technique. When testing a group of sera, negative for anti-HSV antibodies by microneutralization, in the micro-SPRIA, a range of negative reactivities was noted, suggesting that cut-points should be determined for each antigen preparation. The micro-SPRIA detected appropriate antibody activity in patients with recurrent infection and a marked agreement was noted in comparison to detection of anti-HSV antibodies measured with the microneutralization test. The type-specificity of the micro-SPRIA was substantiated by the independence of test results using VP119 and VP123 antigens for a random group of positive sera. The assay is rapid, specific, and sensitive and allows the testing of multiple serum samples with a standardized set of reagents.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

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.

Differentiation markers in oral carcinoma cell lines and tumors.

Cell lines are useful as models if they retain the relevant characteristics of the tissue of origin. We compared two human squamous carcinoma cell lines derived from tumors of the tongue that vary in their extent of differentiation, with human biopsies of carcinomas of the tongue that were either poorly or well-differentiated. The mRNA levels of suprabasal cell proteins (keratin K13, involucrin, transglutaminase) and of protein kinase C (PKC) isozymes were measured by RT-PCR. Apart from PKC beta and PKC delta (mostly expressed by Langerhans cells and missing in culture), qualitatively similar patterns were found in vitro and in vivo. The more differentiated cells had expression levels moderately lower to higher than the normal controls. The poorly differentiated cells generally had substantially lower levels.