Localization of tumor suppressor gene candidates by cytogenetic and short tandem repeat analyses in tumorigenic human bronchial epithelial cells.

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.

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method.

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Measurement of cytochrome P450 2A6 and 2E1 gene expression in primary human bronchial epithelial cells.

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 measurement of many genes simultaneously by quantitative RT-PCR using standardized mixtures of competitive templates.

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.

The gene expression index c-myc x E2F-1/p21 is highly predictive of malignant phenotype in human bronchial epithelial cells.

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.

Loss of spr1 expression measurable by quantitative RT-PCR in human bronchogenic carcinoma cell lines.

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.