Our previous work found DA rats superior for intrinsic aerobic running capacity (ARC) and several cardiac function indexes compared with Copenhagen (COP) rats, and identified ARC quantitative trait loci (QTLs) on rat chromosomes 16 (RNO16) and 3 (RNO3). The purpose of this study was to use these inbred rat strains as a genetic substrate for differential cardiac gene expression to identify candidate genes for the observed ARC QTLs. RNA expression was examined globally in left ventricles of 15-wk-old DA, F1(COP x DA), and COP rats using microarrays to identify candidate genes for ARC QTLs. We identified 199 differentially expressed probe sets and determined their chromosomal locations. Six differentially expressed genes and expressed sequence tags (ESTs) mapped near ARC QTL regions, including PDZ and LIM domain 3 (Pdlim3). Differential expression of these genes/ESTs was confirmed by quantitative RT-PCR. The Ingenuity Pathways program identified 13 biological networks containing 50 (of the 199) differentially expressed probe sets and 85 additional genes. Four of these eighty-five genes mapped near ARC QTL-containing regions, including insulin receptor substrate 2 (Irs2) and acyl-CoA synthetase long-chain family member 1 (Acsl1). Most (148/199) differentially expressed probe sets showed left ventricular expression patterns consistent with the alleles exerting additive effects, i.e., F1(COP x DA) rat RNA expression was intermediate between DA and COP rats. This study identified several potential ARC QTL candidate genes and molecular networks, one of them related to energy expenditure involving Pik3r1 mRNA expression that may, in part, explain the observed strain differences in ARC and cardiac performance.

The metabolic detoxification capacity may critically regulate the susceptibility of human tissues to cancer development. We used standardized and quantitative, reverse transcription‐polymerase chain reaction (StaRT‐PCR) and microarray chip techniques to analyze transcript levels of multiple detoxification enzymes in cultured normal human oral keratinocytes (NOK) and the Siman virus 40 T antigen‐immortalized oral keratinocyte line SVpgC2a, viewing the latter as a model of a benign tumor state. With good agreement between the 2 methodologies, NOK and SVpgC2a were found to express transcripts for cytochrome P450 enzymes (CYPs), factors related to CYP induction and enzymes involved in conjugation reactions or detoxification of reactive oxygen. The cell types expressed similar levels of CYP 2B6/7, CYP 2E1, P450 oxidoreductase, the aryl hydrocarbon receptor nuclear translocator, sulfotransferase 1A1, sulfotransferase 1A3, epoxide hydrolase, glutathione S‐transferase M3, glutathione S‐transferase pi and catalase, superoxide dismutase 1, glutathione peroxidase 1 and glutathione peroxidase 3. In contrast, SVpgC2a exhibited comparatively higher levels of CYP1A1, 1B1, aryl hydrocarbon receptor, glutathione S‐transferase M1, 2, 4, 5, glutathione S‐transferase theta 1 and superoxide dismutase 2 and comparatively lower levels of UDP glycosyltransferase 2 and microsomal glutathione S‐transferase 1. Some transcripts, e.g., CYP 2A6/7, were not detected by either standard, non quantitative RT‐PCR or the above methods, whereas others were barely quantifiable by StaRT‐PCR, i.e., were present at 1–10 molecules/106 molecules of actin. Overall, the expression analysis demonstrated presence of mRNA for multiple enzymes involved in foreign compound metabolism and detoxification pathways, including several enzymes not previously reported for oral epithelium. Generally, the comparison of NOK from 2 individuals indicated relatively similar transcript levels of these enzymes. In contrast, differences between NOK and SVpgC2a, e.g., for CYP1B1, may reflect alteration caused by immortalization and aid identification of early stage tumor markers in oral epithelium. © 2002 Wiley‐Liss, Inc.

Radon exposure is associated with increased risk for bronchogenic carcinoma. Mutagenesis analyses have revealed that radon induces mostly multi-locus chromosome deletions. Based on these findings, it was hypothesized that deletion analysis of multiple radon-induced malignant transformants would reveal common mutations in chromosomal regions containing tumor suppressor genes responsible for malignant transformation. This hypothesis was supported by a previous study in which tumorigenic derivatives of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were established following irradiation with 30 cGy of high linear energy transfer radon-simulated alpha-particles. Herein, we describe the analyses of 10 additional tumorigenic derivative cell lines resulting from the irradiation of five additional independent BEP2D populations. The new transformants have common cytogenetic changes, including the loss of chromosome (ch)Y, one of three copies of ch8, one of two copies of ch11p15-pter and one of three copies of ch14. These changes are the same as those reported previously. Analysis of PCR-amplified short tandem repeats of informative loci confirmed the loss of heterozygosity (LOH) at 12 loci spanning the length of ch8 in cell lines from four of the total of eight irradiation treatments to date and the loss of chY in all cell lines (8 of 8). LOH analysis with a total of 17 informative loci confirmed loss on ch14 in transformants from seven of eight irradiation treatments and indicated a 0.5-1.7 cM region of common involvement centered around locus D14S306. No LOH was detected at any of the informative loci on ch11. The overall results support our stated hypothesis. Further studies are currently in progress to determine whether the ch8 and ch14 regions contain genes with tumor suppressor function in bronchial epithelial cells.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Progress toward complete sequencing of all human genes through the Human Genome Project has already resulted in a need for methods that allow quantitative expression measurement of multiple genes simultaneously. It is increasingly recognized that relative measurement of multiple genes will provide more mechanistic information regarding cell pathophysiology than measurement of individual genes one by one or by methods that do not allow direct intergene comparison. In this study, previously described quantitative reverse transcription-polymerase chain reaction methods were modified in an effort to provide a rapid, simple method for this purpose. Internal standard competitive templates (CTs) were prepared for each gene and were combined in a single solution containing CTs for more than 40 genes at defined concentrations relative to one another. Any subsequent dilution of the CT mixture did not alter the relationship of one CT to another. Because the same CT standard solution or a dilution of it was used in all experiments, data obtained from different experiments were easily compared. The use of multiple CT mixtures with different housekeeping gene to target gene ratios provided a linear dynamic range spanning the range of expression of all genes thus far evaluated. CT stock solutions were used to simultaneously quantify the expression of 25 genes relative to beta-actin and glyceraldehyde-3-phosphate dehydrogenase in normal and malignant bronchial epithelial cells. Because the CT concentrations were known, data in the form of both absolute messenger RNA (mRNA) copy number and mRNA relative to housekeeping gene mRNA were obtained. The methods and reagents described will allow rapid, quantitative measurement of multiple genes simultaneously, using inexpensive and widely available equipment. Furthermore, the CT standard solution may be distributed to other investigators for interlaboratory standardization of experimental conditions.

Bronchogenic carcinomas arise from bronchial epithelial cells (BECs), Inhalation exposure of BECs to nitrosamines in cigarette smoke is an important exogenous risk factor for malignant transformation of BECs. Thus, an important endogenous risk factor is likely to be the capacity of BECs to metabolize nitrosamines, Among the cytochrome P450 enzymes capable of metabolizing nitrosamines, CYP2A6, CYP2E1 and CYP2B6 are expressed in BECs, In this study, we used quantitative RT-PCR to evaluate expression of CYP2A6 and CYP2E1 in primary human BECs from 12 non-smokers and eight smokers. CYP2A6 was expressed in 20/20 cases and quantifiable in 18/20 cases, with a mean level of 580 mRNA/10(6) beta-actin mRNA, CYP2E1 expression was observed in 9/20 cases, but in all cases it was expressed at levels below our limit of quantification (10 mRNA/10(6) beta-actin mRNA), There was significant (P < 0.05) 20-fold inter-individual variation in expression of CYP2A6, Further, the mean level of CYP2A6 among smokers (260 mRNA/10(6) beta-actin mRNA) was significantly lower than among non-smokers (740 mRNA/10(6) beta-actin mRNA). It is hypothesized that: (i) inter-individual variation in CYP2A6 gene expression may contribute to inter-individual variation in risk for bronchogenic carcinoma; (ii) smoking may reduce the level of expression of CYP2A6 in the BECs of some individuals; and (iii) CYP2A6 is more important than CYP2E1 for metabolic activation of nitrosamines in bronchial epithelial cells.

Expression of the small, proline-rich protein (spr1) squamous differentiation marker was measured in five cultured normal and 12 malignant human bronchial epithelial cell (BEC) populations by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Whereas spr1 expression was quantifiable and inducible in all five cultured normal cell populations, in all 12 carcinoma cell lines evaluated it was neither quantifiable nor inducible. Primers spanning the entire spr1 coding sequence amplified full-length PCR product from genomic DNA; therefore, large deletions in the coding region were not responsible for the loss of expression measurable by RT-PCR. This is the first molecular genetic marker reported that distinguishes all normal from all carcinoma cell populations evaluated. Because the spr1 protein is a component of the crosslinked envelope that forms during the squamous differentiation process, we hypothesize that the apparent loss of spr1 gene expression disrupts mechanisms for terminal squamous differentiation in the bronchial epithelium, thereby contributing to malignant transformation.

Recent methodological developments allow expression measurement of many genes simultaneously, thereby revealing patterns of gene expression that can be related to phenotype. We hypothesized that through the use of such methods we could identify patterns of gene expression associated with the malignant phenotype in human bronchial epithelial cells (BEC). To test this hypothesis, a recently developed quantitative reverse transcriptase polymerase chain reaction method was used to assess simultaneously expression of 15 genes mechanistically associated with cell-cycle control (c-myc, E2F-1, p21, rb, PCNA, cyclin D2, cyclin D3, cyclin E, cdc2, CDK2, CDK4, mad, max p21, max p22, and p53) in normal cell cultures from five individuals and in nine different malignant BEC lines. Relative to the mean expression levels in cultured normal cell populations, expression of c-myc, E2F-1, PCNA, cyclin E, and CDK4 messenger RNA (mRNA) were significantly increased and expression of p21 and p53 mRNA were significantly decreased in one or two, but not all three subtypes (squamous, adenocarcinoma and small cell) of carcinoma cell lines evaluated. No single cell-cycle control gene discriminated all three subtypes from normal cell populations. In contrast, the gene expression index c-myc X E2F-1/p21 separated all carcinoma cell lines from all normal cell populations initially evaluated. This malignancy index was validated in an additional three cultured normal BEC and three carcinoma cell lines, as well as three pairs of matched primary normal bronchial epithelial and primary bronchogenic carcinoma samples, and three pairs of matched primary normal lung parenchyma and primary bronchogenic carcinoma tissue. Again, the c-myc X E2F-1/p21 index successfully discriminated all cultured and primary normal from malignant samples and thereby had a predictive value of 1 (no false positives and no false negatives). We hypothesize that because of functional mutations in cell-cycle regulatory genes (e.g., p53 and/or rb), cells lose the ability to maintain a pat tern of gene expression mechanistically associated with normal, division-limited homeostatic equilibrium. Because the c-myc x E2F-1/p21 gene expression index has high specificity for malignant tissue, it will allow confirmation that there is a significant amount of tumor tissue present in small (e.g., fine-needle) biopsy specimens prior to evaluating them for expression of other genes, such as those involved in chemoresistance or radioresistance. In addition, the goal of most gene therapy efforts is to alter levels of gene expression quantitatively. This index and others derived in a similar manner may better define potential gene therapy targets as well as response of targeted genes to therapy.

To establish a cell culture model for lung carcinogenesis, independent populations of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were treated with high linear energy transfer radon-simulated alpha-particles, expanded and xenotransplanted into Nu/Nu mice. Six independent cell lines were established from tumors that developed from three separate radiation treatments as follows: treatment (Tx) 1 (30 cGy--two doses), H2BT, Tx 2 (30 cGy--single dose), R30T1L, R30T2 and R30T3L, Tx 3 (30 cGy--single dose), H1ATN and H1ATBA1. Cytogenetic analysis revealed common changes in all tumor lines: loss of the Y chromosome (ch), one of three copies of ch8, one of three copies of ch14, and one of two copies of ch4p16-pter and ch11p15-pter. Analysis of polymerase chain reaction-amplified short tandem repeats of informative loci confirmed the loss of chY in all lines and loss of heterozygosity (LOH) at eight loci spanning the length of ch8 in all lines from Tx's 1 and 2. Our data support previous studies indicating the presence of tumor suppressor genes on ch8. LOH also was confirmed on ch14 at locus D14S306 in all cell lines from Tx 2 and in one of two lines from Tx 3. This region, 14q12-q13, may contain changes in one of the five known somatostatin receptor genes (SSTR1). No LOH was detected at any of the informative loci tested for on ch4 or ch11.