Biomedical subjects

Gary L Johanning

Publications and source records attributed to Gary L Johanning.

14 recordsLinked to original sources

PubMed2007

Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer.

Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer.

Adenocarcinoma, Clear Cell↗

PubMed2006

Intrinsic cisplatin resistance in lung and ovarian cancer cells propagating in medium acutely depleted of folate.

Many tumors develop intrinsic and/or acquired resistance to cisplatin. The purpose of the present study was to examine the influence of acute extracellular folate depletion prior to cisplatin treatment on the development of intrinsic cisplatin resistance. Lung and ovarian cancer cells were propagated in medium acutely depleted of folate and subsequently treated with cisplatin. The IC50 level for cisplatin, cell viability, cell proliferation, and global DNA methylation were determined. Gene expression profiling was performed using the Atlas Cancer 1.2 Array. Acute extracellular folate depletion resulted in the development of intrinsic cisplatin resistance. Cells propagating in medium acutely depleted of folate had a survival advantage compared to control cells when exposed to cisplatin, and thymidine supplementation did not reverse the intrinsic cisplatin resistance. cDNA microarray analysis revealed some novel genes associated with the development of intrinsic cisplatin resistance. Our report is the first to demonstrate that acute extracellular folate depletion results in intrinsic cisplatin resistance. If these results are confirmed by in vivo human studies, it would suggest that the folate status of the recipient of cisplatin might have an impact on response to that chemotherapeutic agent.

Antineoplastic Agents↗

PubMed2005

Women with polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are less likely to have cervical intraepithelial neoplasia (CIN) 2 or 3.

The role of nutrient-related genetic susceptibility factors for pre-cancerous lesions is gaining attention. We conducted a study to examine associations between polymorphisms in folate pathway coenzymes (methylenetetrahydrofolate reductase [MTHFR] and methionine synthase [MS]) and cervical intraepithelial neoplasia (CIN) 2 or 3 in a population exposed to folic acid by the food fortification program in the United States. Status of MTHFR and MS and circulating concentrations of folate, vitamins B12, A, E, C and total carotene were ascertained in 170 Caucasian and 266 African-American women positive for high-risk human papilloma virus (HR-HPV). Polymorphism status was determined using polymerase chain reaction assays. Micronutrient concentrations were measured using radiobinding assays, high performance liquid chromatography or spectrophotometry. Presence/absence of CIN 2 or 3 was determined on the basis of histology results and the association with risk factors was examined using multivariable analyses. Eighty women had CIN 2 or 3 lesions and they were compared to 356 women who had CIN 1, ASCUS or normal cytology. We found that women polymorphic for MTHFR were less likely to have CIN 2 or 3 (odds ratio [OR] = 0.43, 95% confidence interval [CI] = 0.23-0.79). No associations were seen with MS polymorphism alone (OR = 0.72, 95% CI = 0.43-1.21); however, women polymorphic for both MTHFR and MS were less likely to have CIN 2 or 3 (OR = 0.21, 95% CI = 0.08-0.62). We conclude that these polymorphisms in the folate metabolic pathway were associated with a lower likelihood of CIN 2 or 3 in a population exposed to adequate amounts of folate from exposure to food fortification with folic acid.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2005

Viral detection.

The focus of this chapter is the detection of DNA viruses. The emphasis is on amplification reactions that include reverse transcription-polymerase chain reaction (RT-PCR), PCR, real-time RT-PCR, and real-time PCR methods. Amplification of the E6 and E7 oncoproteins of HPV16 is described in detail, and primers and probes that can be used to amplify these oncogenes are described. Techniques to quantify these oncogenes in infected human tissue specimens are presented, and analysis of data resulting from real-time PCR detection of the E6 and E7 oncogenes is discussed. Other methods for viral nucleic acid detection, including nested PCR amplification, ligase chain reaction, and enzyme-linked immunosorbent assay (ELISA), are also briefly discussed.

DNA Viruses↗

PubMed2004

A novel time-course cDNA microarray analysis method identifies genes associated with the development of cisplatin resistance.

In recent years, most cDNA microarray studies of chemotherapeutic drug resistance have not considered the temporal pattern of gene expression. The objective of this study was to examine systematically changes in gene expression of NCI-H226 and NCI-H2170 lung cancer cells treated weekly with IC10 doses of cisplatin. NCI-H226 lung cancer cells were treated weekly with an IC10 dose of cisplatin. Candidate genes with a fold change of 2.0 or more were identified from this study. A second experiment was conducted by exposing NCI-H2170 cells to cisplatin doses that were increased in week 4 and decreased in week 5. Overall, 44 genes were differentially expressed in both the NCI-H226 and NCI-H2170 cell lines. In the NCI-H2170 cell line, 24 genes had a twofold gene expression change from weeks 3 to 4. Real-time PCR found a significant correlation of the gene expression changes for seven genes of interest. This small time-ordered series identified novel genes associated with cisplatin resistance. This kind of analysis should be viewed as a first step towards building gene-regulatory networks.

Antineoplastic Agents↗

PubMed2004

Micronutrients and cancer therapy.

The effect of micronutrient supplementation on the effectiveness of cancer chemotherapeutic agents is reviewed, and the efficacy of antioxidants, folic acid, and other vitamins and minerals is discussed. Although some micronutrients show promise in enhancing the cytotoxicity of anticancer agents in vitro, caution should be exercised in recommending micronutrient supplementation for cancer patients receiving chemotherapeutic drugs. To date, few well-controlled clinical trials have been conducted to evaluate the efficacy of micronutrients in promoting the sensitivity of tumors to chemotherapeutic agents.

Animals↗

PubMed2003

Detecting the expression of human endogenous retrovirus E envelope transcripts in human prostate adenocarcinoma.

BACKGROUND: The expression of human endogenous retrovirus (HERV) mRNA and proteins was associated recently with diseases that include human malignancies. The authors report that, in the current study, transcripts encoding the envelope region of an HERV family, HERV-E, were expressed in human prostate carcinoma. METHODS: RNA was isolated from various prostate tissues and was tested for the expression of various HERV envelope (env) genes by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, RNA in situ hybridization (ISH), and Northern blot analysis. Variants of HERV that appeared in prostate carcinoma tissues were sequenced, and HERV-E was expressed in prokaryotic and eukaryotic systems. RESULTS: In the current study, the authors found that the mRNA of the env gene of one particular family of HERVs, HERV-E, was expressed in some prostate carcinoma tissues (38.8% positive; n = 49 specimens) but not in normal prostate tissues using RT-PCR, RNA ISH, and Northern blot assays. The expression of HERV-E transcripts in prostate tumor epithelial cells was confirmed further by ISH using an HERV-E specific antisense probe. Approximately 50% of the cDNA of HERV-E obtained from prostate carcinoma specimens contained no stop codon and expressed proteins in prokaryotic or eukaryotic expression systems. Furthermore, the expression of both HERV-E and ERV3 (another class of HERV) was detected in the same prostate carcinoma tissues. CONCLUSIONS: The expression and distribution of multiple HERV-E endogenous retroviral elements in prostate carcinoma, but not in normal control specimens, suggests that they may serve as novel tumor markers for the early diagnosis and immunotherapy of patients with prostate carcinoma.

Blotting, Northern↗

PubMed2003

Quantitation of HERV-K env gene expression and splicing in human breast cancer.

Human endogenous retroviruses (HERVs) comprise up to 8% of the human genome. In previous studies, we demonstrated that type 1 HERV-K envelope (env) transcripts are expressed in most human breast cancers, but not in normal breast tissues. In the current study, we report that type 2 HERV-K env transcripts are also present in human breast cancers. By real-time RT-PCR, the expression of HERV-K env transcripts was 5-10-fold higher in breast cancer cell lines treated with estradiol and progesterone than in cells without treatment, and expression was significantly higher in most breast cancer tissues than in normal breast tissues. Furthermore, both types of HERV-K env transcripts were capable of being spliced into subgenomic env transcripts and various splice donor and acceptor sites were detected in breast cancers. The selective expression and distribution of multiple HERV-K endogenous retroviral element splice variants in breast cancer, but not in normal controls, suggests that they are novel breast tumor markers.

Breast Neoplasms↗

PubMed2003

Retinoids and epigenetic silencing in cancer.

An epigenetic event alters the activity of genes without changing their structure. Methylation reactions are important mediators of epigenetic events in cells. Two recent studies have established a mechanistic link between a vitamin A metabolite and epigenetic alterations of its target promoter that could provide insights about the role of vitamin A in cancer prevention.

Cell Transformation, Neoplastic↗

PubMed2002

Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicolaou tests using real-time polymerase chain reaction analysis.

BACKGROUND: The detection of specific human papillomavirus 16 (HPV-16) E6 and E7 oncogene transcripts may be a sensitive indicator of the direct involvement of viral oncogenes in the development of cervical neoplasia and carcinoma. The goal of this study was to determine the potential clinical uses of real-time polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) methods for evaluating HPV-16 E6 and E7 oncogene expression. METHODS: ThinPrep cervical samples were tested for expression of oncogenes of HPV-16 by real-time PCR or RT-PCR analysis and were compared with detection of expression by conventional PCR and RT-PCR analysis. Both sets of results were correlated with the cytologic diagnosis of the cervical samples. RESULTS: The presence of HPV-16 E6 and E7 DNA and RNA was observed only in HPV-16 positive cervical carcinoma cell lines but not in HPV-18 positive or HPV negative cell lines. The percentage positive for HPV-16 E6 or E7 DNA in a series of ThinPrep cervical cytologic samples (n = 348 samples) was 0% for negative samples (n = 45 samples), 9.7% for atypical squamous cells of undetermined significance (ASCUS; n = 144 samples), 16.9% for low-grade squamous intraepithelial lesion (LSIL; n = 118 samples), and 51.2% for high-grade intraepithelial lesion (HSIL; n = 41 samples). The copy numbers per nanogram for both DNA and RNA E6 and E7 were increased significantly as severity of the lesions progressed from ASCUS to HSIL, and RNA copy numbers were a more sensitive indicator of HPV-16 E6 and E7 expression than DNA copy numbers. The increase in copy numbers took place in a stepwise fashion from ASCUS, to LSIL, to HSIL. CONCLUSIONS: The detection of HPV-16 E6 and E7 expression by real-time RT-PCR or PCR analysis in ThinPrep cervical cytologic specimens may serve as a quick, reliable, and sensitive tool to identify a subset of patients who express HPV-16 oncoproteins.

Adolescent↗

PubMed2002

Effects of energy expenditure and Ucp1 on photoperiod-induced weight gain in collard lemmings.

OBJECTIVE: To determine the role of total energy expenditure (TEE) and its components in the ability of collared lemmings to increase weight in response to a decrease in photoperiod. RESEARCH METHODS AND PROCEDURES: Energy expenditure was measured by 24-hour indirect calorimetry concurrent with food-intake studies. TEE and resting and nonresting energy expenditure (REE and NREE, respectively) were adjusted for body weight by analysis of covariance (ANCOVA). Uncoupling protein 1 (Ucp1) mRNA levels from interscapular brown adipose tissue were determined by Northern blot. RESULTS: TEE and REE of lemmings exposed to a short photoperiod for 10 days were significantly lower than that of lemmings exposed to a long photoperiod (p < 0.05), whereas NREE was not significantly different (p = 0.44). Ucp1 mRNA levels in interscapular brown adipose tissue were 50% lower in short- vs. long-photoperiod lemmings (p < 0.01). Ucp1 mRNA levels were positively related to REE (r2 = 0.79, p < 0.01). After adjustment of REE for differences in Ucp1 mRNA levels, there was no longer a significant difference attributable to photoperiod treatment (p = 0.54). DISCUSSION: The results of this study indicate that the increase in body mass that occurs when collared lemmings are exposed to a short photoperiod may be primarily fueled by a decrease in REE and is correlated with a decrease in Ucp1 mRNA levels.

Adipose Tissue, Brown↗

PubMed2002

DNA methylation and diet in cancer.

The studies reviewed here investigate the association between folate status and DNA methylation in cancer tissues. We evaluated tissue vitamin levels and global DNA methylation, a biomarker of neoplasia, in normal lung and lung cancer tissues. Lung squamous cell carcinoma tissues exhibited global DNA hypomethylation, with decreased folate and vitamin B-12 concentrations, and increased vitamin C concentrations, relative to matched uninvolved control tissues. Breast cancer tissues also had globally hypomethylated DNA and decreased vitamin B-12 and vitamin C levels, but folate concentrations were elevated in breast cancer tissues. Global DNA methylation status in buccal mucosal cells may reflect global methylation status in lung tissues, because there was a significant association between global DNA methylation in buccal mucosal cells and malignant tissues of the lung, but not between methylation in peripheral leukocytes and lung tissues. We found that global DNA hypomethylation, as assessed by a radiolabeled 5-methylcytosine technique, was associated with susceptibility for development of lung cancer, which is involved in the progression of the disease. DNA methylation was also associated with the development of squamous cell carcinomas in whites but not in blacks. Overall, these studies suggest that global DNA methylation patterns may vary depending on the type of cancer, that tissue vitamin levels are associated with global DNA methylation status and that ethnicity should be considered in studies of DNA methylation.

Age Factors↗

PubMed2002

Cellular vitamins, DNA methylation and cancer risk.

The overall goal of this research is to evaluate interactions among cellular vitamin levels and global DNA hypomethylation and the impact of these variables on human cancer risk. Global DNA methylation was determined by two methods: a radiolabeled methyl incorporation (RMI) assay and an immunohistochemical assay using an antibody to 5-methylcytosine (5-MC). The RMI assay is useful for evaluating methylation of DNA in tissue samples, whereas the 5-MC assay clearly reveals DNA methylation in specific types of cells and has minimal day-to-day variability. We have observed significant interactions among cancer-protective vitamins and global DNA methylation at the level of tissues. A significant positive association was observed between global DNA methylation in buccal mucosal cells and malignant tissues, but not between global DNA methylation in peripheral leukocytes and malignant tissues of the lung. These results suggest that changes in global methylation in buccal mucosal cells may reflect changes in tissues at high risk of developing lung cancer. With the antibody technique, we have demonstrated that alterations in global DNA methylation are associated with epigenetic differences in susceptibility for development of lung cancer, which is involved in the progression of the disease. The effect of race on these relationships also is discussed. Significant associations observed between expression of epidermal growth factor receptor and global DNA methylation, as assessed by the 5-MC assay but not by the RMI assay, indicate that evaluation of global methylation and biomarkers in specific types of cells may shed light on the associations between global DNA methylation and other intermediate endpoint biomarkers in the future.

5-Methylcytosine↗

PubMed2002

Effects of body weight gain reduction resulting from chemopreventive agent treatment on mammary gland morphology.

Moderate reductions (< or = 15%) in body weight gain, similar to those observed after administration of some chemopreventive agents in chemically induced mammary cancer models, will result in decreased mammary cancers (up to 55%). The objective of this study was to determine whether changes in mammary gland differentiation, proliferation, apoptosis, and estradiol and progesterone levels are affected by moderate reductions in body weight induced after chemopreventive agent treatment and dietary restriction. The body weights of female Sprague-Dawley rats were reduced by dietary restrictions to match those of rats receiving 4-hydroxyphenylretinamide (4-HPR) at a dose known to inhibit methylnitrosourea (MNU)-induced mammary cancers. 4-HPR supplementation or dietary restrictions began 1 wk before MNU administration at 50 days of age. Mammary gland differentiation, proliferation, apoptosis, and serum levels of estradiol and progesterone were measured at 50, 57, and 71 days of age. Casein expression, proliferating cellular nuclear antigen expression, and apoptosis were not significantly different from controls in the dietary-restricted group. Proliferating cellular nuclear antigen expression was significantly lower in 4-HPR-treated animals than in controls at 57 days of age. The diameter of the mammary gland ducts was smaller at 71 days of age in the treatment groups. A decrease in estradiol levels for each group was observed at 50 days of age, but not at later time points. Progesterone levels were reduced in the 4-HPR group, but not in the dietary-restricted group, during each time period. It would appear that the observed decrease in mammary cancers observed with moderate reductions in body weight gain might be due to multiple related factors different from those related to 4-HPR treatment.

Alkylating Agents↗