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Kenneth R Shroyer

Publications and source records attributed to Kenneth R Shroyer.

32 records · Page 2Linked to original sources
PubMed2003

Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells.

Bladder cancer is the fourth and eighth most common cancer in men and women in the United States, respectively. Survivin, a member of inhibitor of apoptosis protein (IAP) gene family, is deregulated in a wide range of malignancies, including carcinoma of the bladder urothelium. Recent advances have identified survivin as a novel intervention target to induce apoptosis in cancer cells by phytochemicals or synthetic agents. Silibinin is a naturally occurring flavanone, isolated from milk thistle extract, and has been shown to possess cancer prevention/intervention potential against various cancers. In several animal and human studies, it is found to be safe and non-toxic. Human bladder transitional-cell papilloma RT4 cells were treated with silibinin and analyzed for survivin protein and mRNA levels by Western blotting and real-time RT-PCR, respectively. Silibinin treatment of cells for 24 h at 100 microM dose resulted in approximately 50% decrease in survivin protein level; however, treatment at 200 microM dose for 24 and 48 h showed a complete loss in survivin protein without any change in actin used as loading control. Employing RT-PCR analysis we also observed that silibinin causes a strong to complete decrease in survivin mRNA levels. In other studies, down-regulation of survivin by silibinin was associated with a very strong and prominent caspases-9 and -3 activation as well as PARP cleavage. Quantitative apoptotic assay showed that silibinin decreased survivin levels and caspases-PARP cleavages, in accord with a strong apoptotic death and growth inhibition of RT4 cells. Together, these findings suggest that more studies are needed to investigate in vivo effect of silibinin on survivin expression and associated biological effects in bladder cancer that could provide useful information for silibinin efficacy in the prevention/intervention of human bladder cancer.

Apoptosis↗
PubMed2003

Innovations in understanding the biology of cervical cancer.

Revelation of the connection between the human papillomavirus (HPV) and cervical neoplasia and invasive cervical cancer is prompting new investigations to expand that understanding and promote vaccines, gene therapy, and other interventions. At the Second International Conference on Cervical Cancer (Houston, TX, April 11-14, 2002), laboratory and clinical researchers reported advances in new studies meant to increase understanding of the natural history of HPV and cervical intraepithelial neoplasia, to evaluate new cervical cancer screening techniques, and to promote new therapies. Using K14-HPV type 16 transgenic mice, researchers are investigating the effects of estrogen on cervical cancer carcinogenesis, and results are lending support to epidemiological theories showing a difference in HPV infection rates and the development of cervical lesions in women using oral contraceptives. Other work involves investigating genes that are up-regulated by HPV infection and the role of the p53 homologue, p63, in cervical neoplasia evolution. Telomerase also is under investigation as a biomarker in high-risk populations. Gene therapy that replaced p53 in cervical cancer cell lines in vitro and a nude mouse model inhibited cell and tumor growth, confirming previous findings in squamous epithelial carcinomas of the head and neck. Furthermore, research in intracellular targeting of antigens to subcellular locations shows promise for treating cervical cancer preclinically. Identification of molecular changes in cervical cancer and knowledge about the importance of HPV infection in cervical cancer can lead to new therapies to treat existing cervical cancer and, in the long term, prevent the disease.

Animals↗
PubMed2003

Detection of occult cervical micrometastases in patients with head and neck squamous cell cancer.

OBJECTIVE: The incidence of occult nodal metastases associated with head and neck squamous cell carcinoma (HNSCC) and the clinical significance of nodal micrometastases by cytokeratin immunohistochemical analysis are examined. STUDY DESIGN: In all, 1012 lymph nodes from 50 patients treated between 1992 and 2001 at the University of Colorado Health Sciences Center (Denver, CO) were evaluated retrospectively for micrometastases. METHODS: Serial sectioning in 5-to 6-microm interval specimens stained either with hematoxylin and eosin (H&E) or immunostaining for cytokeratins using the monoclonal antibody cocktail AE1/AE3 was performed in 21 N0, 11 N1, and 14 N2 patient cases. Cases that showed scattered cells with suspect staining qualities but without morphological features consistent with HNSCC were further evaluated by epithelial membrane antigen (EMA) immunohistochemical analysis. RESULTS: H&E-stained and cytokeratin-stained sections revealed occult nodal micrometastases in 3.8% of N0 and 5% of N1 cases. Overall, 26 micrometastases were identified in N0 and N1 patients, causing 29% of N0 patients and 45% of N1 patients to be upstaged. Cytokeratin immunostaining detected micrometastases in eight cases that were negative on H&E serial sectioning. Serial sectioning by H&E alone identified three additional micrometastases. Negative EMA immunostaining confirmed the absence of malignant cells in lymph node sections that were equivocal on cytokeratin staining. CONCLUSIONS: The use of serial sectioning with H&E and cytokeratin immunohistochemical analysis increases the detection of micrometastases that are often elusive by routine processing in patients with HNSCC. Improved methods of detecting micrometastases may provide a basis for improved planning of postoperative therapy for patients already at risk for tumor recurrence.

Antibodies, Monoclonal↗
PubMed2003

Monoclonal expansion with integration of high-risk type human papillomaviruses is an initial step for cervical carcinogenesis: association of clonal status and human papillomavirus infection with clinical outcome in cervical intraepithelial neoplasia.

To define the natural history of cervical intraepithelial neoplasia (CIN) as related to clonal status, we evaluated 20 cases of CIN1 and 18 cases of CIN2 that had been clinically followed for 7 to 48 months at Osaka University Hospital. These included 10 cases that progressed, 15 cases that persisted, and 13 cases that regressed. We analyzed the clonal status of each case by analysis of the pattern of X-chromosomal inactivation. Human papillomavirus (HPV) infection was detected by PCR-RFLP analysis. CINs that are monoclonal or infected by high-risk HPVs are more likely to progress or persist than cases that are polyclonal or negative for high-risk HPVs (p = 0.009 for monoclonal vs polyclonal, p = 0.024 for high-risk HPV positive vs negative p = 0.024). Eighteen (90%) of 20 monoclonal, high-risk HPV-associated CINs progressed or persisted, whereas 9 (60%) of 15 polyclonal or high-risk HPV-negative CINs regressed. Therefore, the combination of clonality status and high-risk type HPV infection was significantly correlated with clinical outcome (p = 0.003). The physical status of the HPV genome was evaluated in 17 cases of HPV-16 positive CINs by real-time PCR. Of those, the HPV viral genome was present in both episomal and integrated forms in 14 CINs (84%), and 12 of these cases (86%) were monoclonal in composition. By contrast, all three CINs in which the HPV genome was present in episomal form were polyclonal. In one CIN1 that was polyclonal, HPV-16 was originally present in episomal form but after 24 months, the patient developed a monoclonal CIN3 in which the HPV-16 genome was present in mixed form. These results may imply that HPV viral integration into the host genomic DNA is associated with progression from polyclonal to monoclonal status in CIN. These events may play a fundamental role in the progression from low-grade to higher grade dysplasia of the cervical mucosa.

Clone Cells↗
PubMed2003

Survivin in glioblastomas. Protein and messenger RNA expression and comparison with telomerase levels.

CONTEXT: Survivin is a novel inhibitor of apoptosis that acts via a pathway independent of bcl-2. Little is known about its distribution in brain tumors or how it correlates with other biomarkers of malignancy, such as telomerase, an enzyme that plays a critical role in cellular immortalization and cancer biology. OBJECTIVES: To assess survivin protein expression in gliomas and to compare expression with that of telomerase. DESIGN: Immunohistochemical staining for survivin protein expression was performed using an antibody developed in our laboratory. Quantitative survivin messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction. In selected cases, survivin results were compared with quantitative telomerase values analyzed by polymerase chain reaction-based telomerase repeat amplification protocol (TRAP) assay. Twenty-five tumor tissue samples from 16 cases of glioblastoma multiforme (GBM; including multiple tissue samples in 6 patients), 2 grade II gliomas, 4 grade III gliomas, and 3 control temporal lobectomy specimens were studied. RESULTS: Nuclear immunoreactivity for survivin protein and survivin mRNA were detectable in most glioma samples, regardless of grade. Glioblastoma multiforme demonstrated moderate protein expression and survivin mRNA levels compared to epithelial malignancies previously tested in our laboratory. Although the association of survivin mRNA with the levels of telomerase within the GBM cases did not reach statistical significance, most GBMs also expressed survivin. The quantitative score for survivin mRNA was higher in GBMs than in grade II and III gliomas (P =.02), after accounting for multiple specimens per patient. CONCLUSIONS: Quantitative survivin mRNA analysis, but not immunohistochemistry, distinguished GBMs from lower grade gliomas. Mechanisms that promote both cell proliferation (telomerase expression) and cell survival (survivin expression) are often activated in GBMs.

Adult↗
PubMed2003

Sputum cytological atypia as a predictor of incident lung cancer in a cohort of heavy smokers with airflow obstruction.

Individuals with cytological atypia in sputum may be at increased risk for lung cancer. We conducted a longitudinal analysis of the association between lung cancer incidence and cytological atypia in sputum samples collected prospectively from an ongoing cohort of adults at high risk for lung cancer. Cohort members had a smoking history of > or = 30 pack-years and chronic obstructive pulmonary disease documented by pulmonary airflow testing. Sputum samples collected at baseline and periodically thereafter were examined by standard cytological methods. From the cohort of 2,006 people, there were 83 incident lung cancers over 4,469 person-years of observation. At baseline, the association between personal and behavioral characteristics, and sputum cytological atypia was assessed by multiple logistic regression. The association between sputum cytological atypia and incident lung cancer was then assessed by hazard ratios using proportional hazards regression analysis, adjusting for potential confounding factors. Cytological atypia graded as moderate or worse was associated with continuing cigarette smoking (adjusted odds ratio, 2.5; 95% confidence interval, 1.5-4.1), and with lower levels of intake of fruits and vegetables (P for trend = 0.04). Atypia was not associated with several other factors, including the degree of airflow obstruction, the use of vitamin supplements, nonsteroidal anti-inflammatory drugs, or metered-dose steroid inhalers. Incident lung cancer was increased among those with moderate or worse cytological atypia (adjusted hazards ratio, 2.8; 95% confidence interval, 1.4-5.5). This association was not confounded by other risk factors. We conclude that in this high-risk cohort, cytological atypia is associated with continuing smoking and low intake of fruits and vegetables, but that independent of these and other factors, the risk of incident lung cancer is increased among those with moderate or worse grades of cytological atypia in their sputum.

Adult↗
PubMed2002

Analysis of telomerase as a diagnostic biomarker of cervical dysplasia and carcinoma.

Telomerase expression is a potentially important marker of high-grade cervical dysplasia and squamous cell carcinoma (SCC). The routine practice of cervical cytology is limited by problems of false negative diagnoses as well as by poor specificity for clinically significant lesions in patients with low-grade cytologic abnormalities. Telomerase is widely expressed in most SCCs as well as in a high proportion of high-grade squamous intraepithelial lesions. Histochemical studies have confirmed that telomerase is expressed in the lower portions of normal or metaplastic squamous mucosa but that telomerase positive cells extend into the upper epithelial layers in cases of high-grade dysplasia. Since the cervical smear samples the uppermost cell layers of the cervical mucosa, but does not normally include cells derived from the lower layers of the squamous mucosa, the detection of telomerase in exfoliated cells of the cervical smear may have specificity for clinically significant lesions. The analysis of hTR, hTERT, and telomerase activity are complicated by a number of technical factors that may lead to either false negative or false positive test results. Thus, the practical application of telomerase analysis as a diagnostic adjunct for cervical cytopathology may depend on the development of more reliable and sensitive assay systems, possibly formatted for cytochemical applications.

Biomarkers↗
PubMed2002

Quantitative analysis of telomerase hTERT mRNA and telomerase activity in endometrioid adenocarcinoma and in normal endometrium.

OBJECTIVES: In the current study, the quantitative levels of telomerase hTERT mRNA and the functional telomerase repeat amplification protocol (TRAP) assay were correlated with tumor grade in endometrial carcinomas and with the histologic phase of normal endometrium. METHODS: Twenty-six samples of endometroid adenocarcinoma and 20 cases of benign endometrium were obtained from hysterectomy specimens. Total RNA was extracted from each tissue sample and used for quantitative real-time RT-PCR of hTERT mRNA and the levels were standardized to the levels of ribosomal RNA. Quantitative determination of telomerase activity was performed by the polymerase chain-based TRAP assay and the levels of expression were defined by the ratio of radioactivity incorporated into the 6-bp telomerase amplification products versus the radioactivity incorporated into an internal standard (telomerase/ITAS x 100 = 1 RU). Statistical analyses were performed using the Fisher exact test or chi2 test, a Wilcoxon rank sum test, and a linear regression analysis. RESULTS: hTERT mRNA and telomerase activity levels showed a linear association in the study group (P = 0.006, R2 = 0.139). hTERT mRNA levels and telomerase activity levels were significantly higher in endometrial cancer (179 pg/ng rRNA, 44 relative units (RU)) than in normal endometrium (45 pg/ng), (15 RU) (P = 0.009, P = 0.006). In normal endometrium, hTERT mRNA and telomerase activity levels were highest in the proliferative phase (74 pg/ng rRNA, 25 RU) and were relatively low in secretory (13 pg/ng rRNA, 6 RU) and atrophic endometrium (9 pg/ng rRNA, 2 RU). CONCLUSION: These results suggest that the quantitative analysis of hTERT and telomerase activity may have potential roles as diagnostic or prognostic adjuncts for both premenopausal and postmenopausal patients with endometrial cancer.

Adult↗
PubMed2002

Correlation of survivin mRNA detection with histologic diagnosis in normal endometrium and endometrial carcinoma.

BACKGROUND: The objective of this study was to determine if survivin mRNA expression is a marker of endometrioid adenocarcinoma and if survivin mRNA levels correlate with tumor grade and stage. METHODS: Twenty-six samples of endometrioid adenocarcinoma and 18 cases of benign endometrium were obtained at surgery. RNA was extracted from tissues and was used for quantitative real-time RT-PCR, targeting a 91-bp sequence of survivin mRNA and the levels were standardized to the levels of ribosomal RNA. Statistical analysis of the correlation between histologic diagnosis and the corrected survivin mRNA levels was performed by the Fisher Exact test and the Kruskal-Wallis test. RESULTS: Survivin mRNA was detected in all specimens. Survivin mRNA levels were increased in proliferative endometrium (P = 0.0509) and was increased in correlation with ascending grade in endometrioid adenocarcinoma (P = 0.01). CONCLUSION: Survivin mRNA is not a specific marker of endometrial cancer, but may reflect an important mechanism in tumor progression of the endometrial mucosa.

Adenocarcinoma↗
PubMed2002

Clonal analysis and mutations in the PTEN and the K-ras genes in endometrial hyperplasia.

The patterns of X chromosome inactivation and mutations of PTEN and K-ras were evaluated in cases of endometrial hyperplasia to determine the presence of potentially premalignant neoplastic versus polyclonal benign cell populations. Endometrial glandular epithelial cells were collected by laser capture microdissection, and genomic DNAs were extracted. Following treatment with the methylation sensitive restriction endonuclease Hha I, polymerase chain reaction amplification was performed targeting a highly polymorphic short tandem repeat of the human androgen receptor gene (HUMARA). PTEN and K-ras gene mutations were evaluated by analysis of single-strand conformation polymorphism. Two pathologists performed histologic diagnosis of the lesions independently. Monoclonal composition was demonstrated in 13 of 15 (87%) endometrial hyperplasias with atypia and 17 of 31 (55%) complex hyperplasias without atypia. Cytological atypia is significantly associated with the clonal status of the endometrial hyperplasia (13/15 vs 17/31, P = 0.049). In contrast, all 14 normal endometrial tissue samples were polyclonal. PTEN gene mutations were detected in 4 of 13 (30%) monoclonal endometrial hyperplasias with atypia and 2 of 17 (12%) monoclonal endometrial hyperplasias without atypia but were not detected in polyclonal endometrial hyperplasias, with or without atypia. K-ras gene mutations were present in 3 of 13 (23%) monoclonal endometrial hyperplasias with atypia but not in 2 cases of polyclonal endometrial hyperplasia with atypia or in 26 cases of endometrial hyperplasia without atypia. K-ras mutation is thus significantly more frequently found in endometrial hyperplasias with atypia than those without atypia (3/15 vs 0/31, P = 0.030). This study indicates that most cases of endometrial hyperplasia with atypia and a high proportion of cases of endometrial hyperplasias without atypia originate from a single progenitor cell, possibly as a result of genetic alterations, rather than as a result of benign reactive processes.

Cell Transformation, Neoplastic↗
PubMed2002

Immunohistochemical localization of the IAP protein survivin in bladder mucosa and transitional cell carcinoma.

In the current study, the immunohistochemical localization of survivin was correlated with the histologic diagnosis in healthy transitional cell epithelium, transitional cell carcinoma (TCC) in situ, and TCC of the urinary bladder. Forty-five TCCs (grades 1-3, 15 of each), 14 cases of TCC in situ, and II healthy bladder mucosal specimens were selected from archival collections of formalin-fixed bladder tissues. Slides were reacted with an anti-survivin antibody using a conventional immunohistochemical method and then were scored for nuclear and cytoplasmic staining. Statistical analysis of survivin expression was performed to evaluate the correlation of staining pattern with histologic diagnosis and clinical outcome. Nuclear staining for survivin was detected in 26 of 45 TCCs and in 2 of 14 cases of TCC in situ, but was not detected in healthy bladder mucosa. The association of nuclear staining with TCC versus both healthy bladder mucosa and TCC in situ was statistically significant (P < 0.001). Patients with TCC and a nuclear pattern of survivin localization had a greater period of disease-free survival (27.2 months) than was observed in patients with TCC that showed no nuclear staining for survivin (9.9 months); however, the differences were not statistically significant. Nuclear localization of survivin is a marker of TCC but is rarely present in premalignant or benign bladder mucosal specimens.

Amino Acid Sequence↗
PubMed2002

Immunohistochemical localization of survivin in benign cervical mucosa, cervical dysplasia, and invasive squamous cell carcinoma.

Survivin is an inhibitor of apoptosis protein (IAP) that is expressed in fetal development and in cancer Survivin expression in premalignant lesions remains undefined. We obtained 73 samples of cervical squamous tissue, including 31 normal, 17 low- and 15 high-grade squamous intraepithelial lesions (LSILs, HSILs), and 10 squamous cell carcinomas (SCCs)from cone biopsy and hysterectomy specimens, and stained for survivin using an immunoperoxidase method. Nuclear staining was detected in normal mucosa, LSILs, and HSILs; staining intensity was greatest in cases with morphologic evidence of human papillomavirus (HPV) infection. In situ hybridization of serial sections demonstrated colocalization of HPV DNA and survivin. Cytoplasmic staining was observed in immature squamous metaplasia and in SCCs. Survivin expression in immature metaplastic squamous mucosa may reflect a rolefor survivin in normal squamous differentiation. However, the histologic correlation between nuclear staining and HPV infection suggests involvement of survivin in HPV-mediated disruption of normal cellular maturation.

Animals↗
PubMed2002

Immunohistochemical localization of cdc6 in squamous and glandular neoplasia of the uterine cervix.

CONTEXT: Cdc6 has been extensively studied as a marker for cellular proliferation that is expressed during the normal cell cycle. Recent studies indicate that Cdc6 may be a marker for cervical intraepithelial neoplasia (CIN) and carcinoma; however, the histologic distribution of Cdc6 has not been explicitly defined. Expression of Cdc6 in the endocervical mucosa also remains unexplored. OBJECTIVE: The goal of the current study was to evaluate the distribution of Cdc6 protein, MIB-1 protein, and human papillomavirus (HPV) DNA in a broad range of cervical tissues, including normal, potentially premalignant, and malignant lesions of the ectocervical and endocervical mucosa. METHODS: We used an indirect immunoperoxidase method to stain formalin-fixed, paraffin-embedded tissues and frozen tissues, including biopsy and hysterectomy specimens, for Cdc6 and MIB-1 proteins, and we used in situ hybridization to detect HPV DNA in a subset of cases. RESULTS: Cdc6 staining was exclusively nuclear and was present in both squamous and glandular epithelial cells of histologic sections. Cdc6 staining was rarely present in specimens of normal cervical squamous mucosa (2/84, 2.4%) or in specimens with squamous metaplasia (3/59, 5.1%) and was not detected in normal endocervical glands (0/84). Staining was present in most cases of CIN I (31/48, 65%). Staining was present in the majority of cases of CIN II (25/28, 89%) and in all cases of CIN III (36/36) and squamous cell carcinomas (34/34). The proportion of cells staining for Cdc6 increased with the grade of dysplasia, and the proportion of stained cells in squamous cell carcinomas was similar to that in lesions of high-grade dysplasia. Cdc6 staining was present in the majority of cases in glandular lesions including adenocarcinoma in situ (11/14, 79%) and adenocarcinoma (8/10, 80%). The histologic distribution of Cdc6-immunoreactive cells was similar to that of cells with a strong signal for HPV DNA, but Cdc6 protein and HPV DNA did not colocalize at the level of individual cells. CONCLUSION: Cdc6 expression is a marker for high-grade cervical squamous and glandular dysplasia and carcinoma and is associated with HPV infection. The mechanistic basis of the association between HPV infection and Cdc6 immunopositivity remains to be determined but may represent either up-regulation of Cdc6 expression or stabilization of the Cdc6 protein.

Adenocarcinoma↗