Differential gene expression in chemically induced mouse lung adenomas

Abstract

Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS) was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60%) of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, alpha-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK), alpha-adaptin, alpha-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and alpha- and beta-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240) and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of lung carcinogenesis and in developing biomarkers for lung cancer chemoprevention studies in mice.

Figure 1

Histology of a normal lung and a lung adenoma used in this study. Light photomicrographs of a normal lung (A and B) and a lung adenoma (C and D) at x4 and x40 magnifications, respectively.

Figure 2

Analysis of differentially expressed genes in mouse lung adenomas using CCLS. (A) Schematic illustration of CCLS. Equal amounts of total RNA from lung tumors and normal tissues were converted to cDNA probes with incorporation of ³²P into the cDNA strands during RT. Two competitors were also generated from normal lung tissues using the same procedure except for ³²P incorporation. Probes 1 and 2 were used to perform differential hybridization against a mouse lung cDNA library. (B) An example of data from CCLS. Two identical filters were differentially hybridized with the cDNA probes. The left one represents hybridization with the probe generated from normal tissue, whereas the right one represents hybridization with the probe derived from a lung tumor. The three spots indicated by arrows show three differentially expressed clones that were identified by CCLS. (C) Results of dot blot analysis. Dot blot analysis was conducted as one confirmation step. Differentially expressed clones selected from CCLS were confirmed by dot blot analysis. The GAPDH was used as an internal control.

Figure 3

RT-PCR verification of differentially expressed genes detected using CCLS. “N” represents the normal mouse lung tissue; “T” represents the MNU-induced mouse lung adenomas. GAPDH was applied as an internal control to determine the amount of template in each reaction. (A) RT-PCR confirmation of upregulated genes. Zfp96, zinc finger protein 96; CHUK, conserved helix-loop-helix ubiquitous kinase; SP-A, surfactant protein A; and JAK1 protein. (B) RT-PCR confirmation of downregulated genes. Cry61, growth factor-inducible immediate early gene; CC10, Clara cell protein 10; Emb11, 11-day embryo cDNA; and CA IV, carbonic anhydrase IV.

Figure 4

Characterizations of FLJ11240 hypothetical protein, and neuronal guanine nucleotide exchange factor. (A) The sequence alignments of FLJ11240 hypothetical protein and neuronal guanine nucleotide exchange factor (Ngef) with human counterpart proteins. (B) RT-PCR verification of differential expressions of FLJ11240 hypothetical protein and neuronal guanine nucleotide exchange factor. “N” represents the normal mouse lung tissue; “T” represents the MNU-induced mouse lung adenomas. GAPDH was applied as an internal control to determine the amount of template in each reaction.