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.

Background: Although 40-50% of non-small cell lung cancer (NSCLC) tumors respond to cisplatin chemotherapy, there currently is no way to prospectively identify potential responders. The purpose of this study was to determine whether transcript abundance (TA) levels of twelve selected DNA repair or multi-drug resistance genes (LIG1, ERCC2, ERCC3, DDIT3, ABCC1, ABCC4, ABCC5, ABCC10, GTF2H2, XPA, XPC and XRCC1) were associated with cisplatin chemoresistance and could therefore contribute to the development of a predictive marker. Standardized RT (StaRT)-PCR, was employed to assess these genes in a set of NSCLC cell lines with a previously published range of sensitivity to cisplatin. Data were obtained in the form of target gene molecules relative to 10 6 β-actin (ACTB) molecules. To cancel the effect of ACTB variation among the different cell lines individual gene expression values were incorporated into ratios of one gene to another. Each two-gene ratio was compared as a single variable to chemoresistance for each of eight NSCLC cell lines using multiple regression. In an effort to validate these results, six additional lines then were evaluated.

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH 4 , under oxidative stress. Importantly, pharmacological deprivation of NO in prepubertal to pubertal animals stiffens the extracellular matrix and induces precancerous lesions in the mammary tissues. These lesions overexpress a fibrogenic cytokine, TGFβ, and an oncogene, ERBB2, accompanied by the occurrence of senescence and stem cell-like phenotype. Consistently, normalization of NO levels in precancerous and cancerous breast cells downmodulates TGFβ and ERBB2 and ameliorates their proliferative phenotype. This study sheds new light on the etiological basis of precancerous breast lesions and their potential prevention by manipulating the basal NO level.

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 pattern 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.

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.