Amplification and overexpression of the cyclin D1 and epidermal growth factor receptor genes in non-small-cell lung cancer. Lung Cancer Study Group.

PURPOSE: To study the structure and expression of the cyclin D1 and the epidermal growth factor receptor (EGFR) genes in a cohort of 298 non-small-cell lung cancer (NSCLC) specimens. METHODS: Gene structure was studied by Southern analysis, and gene expression was studied by Northern analysis and immunohistochemical analysis. RESULTS: Amplification of the cyclin D1 locus was found in 14/298 (5%) specimens. All 12/12 specimens with amplification of the cyclin D1 gene for which RNA was available were found to express the cyclin D1 transcript, and 11/12 overexpressed the transcript to levels higher than that of uninvolved lung. The EGFR gene was amplified in 17/286 samples of NSCLC tested, and was overexpressed in 22/169 (13%) cases tested, including 12/13 cases with amplification of the gene for which RNA was available. Cyclin D1 gene amplification was associated with advanced lymph node involvement (P = 0.043), but not with larger tumor size or adverse outcome. Cyclin D1 gene amplification and overexpression occurred independently of retinoblastoma tumor-suppressor gene (RB) inactivation, but tumors with amplification of the cyclin D1 gene were more likely to have EGFR gene amplification (P < 0.005). No correlation of EGFR gene amplification or overexpression with tumor size, lymph node involvement, patient demographic data, or survival was identified. CONCLUSIONS: These data indicate that the cyclin D1 and EGFR genes are amplified and overexpressed in NSCLC, and amplification of the cyclin D1 gene occurs frequently in conjunction with amplification of the EGFR gene.

Prk, a cytokine-inducible human protein serine/threonine kinase whose expression appears to be down-regulated in lung carcinomas.

We have cloned and characterized a putative protein serine/threonine kinase termed prk through a combination of polymerase chain reaction and conventional cDNA library screening approaches. There are apparently two distinct domains within prk protein deduced from its nucleotide sequences. The amino-terminal portion has the feature of the catalytic domain of a serine/threonine kinase and shows strong homology to mouse fnk and other polo family kinases including mouse snk, human and murine plk, Drosophila polo, and yeast Cdc5. The carboxyl-terminal portion, presumably the regulatory domain, shares extensive homology to mouse fnk. Northern blotting analyses reveal that prk expression is restricted to a very limited number of tissues with placenta, ovaries, and lung containing detectable amounts of prk mRNA. prk mRNA expression is also detected at a low level in the megakaryocytic cell line Dami, MO7e, and three brain glioma cell lines. In addition, refeeding of serum-deprived MO7e, Dami, and K562 cells of hematopoietic origin and GMOO637D of lung fibroblasts rapidly activates prk mRNA expression with its peak induction around 2 h after serum addition. prk gene activation by the serum requires no new protein synthesis. The recombinant cytokines such as interleukin-3 and thrombopoietin also activate prk mRNA expression in MO7e cells. Furthermore, a survey of RNAs isolated from the tumor and the uninvolved tissues from 18 lung cancer patients reveals that prk mRNA expression is significantly down-regulated in tumor tissues. Southern blotting analysis indicates that the prk gene is present in a single copy in the genome of tumors and normal cells. Taken together, these results suggest that prk expression may be restricted to proliferating cells and involved in the regulation of cell cycle progression. The molecular cloning of prk cDNA will facilitate the study of its biological role as well as its potential role in tumorigenesis.

Expression patterns of immediate early transcription factors in human non-small cell lung cancer. The Lung Cancer Study Group.

In 1995, there will be 172,000 new cases of lung cancer diagnosed and 153,000 deaths from this disease in the United States. While the pathogenesis of the disease process is poorly understood, a growing body of evidence suggests that abnormalities in cellular regulatory genes may play an important role in the induction, maintenance and/or progression of some tumor types. These genes include both growth promoting oncogenes as well as growth inhibitory or suppressor genes. Included among these genetic sequences are several cellular transcription factors. A group of these factors including c-jun, c-fos and EGR1 are members of a class of genes known as immediate early genes whose expression are inducible by a variety of stimuli including mitogenic and differentiation inducing growth factors, indicating a potential important role for these genes in normal growth processes. Since these genes are involved in early regulation of cellular growth properties and at least two (c-jun and c-fos) can act as oncogenes, we wished to determine whether their expression levels were altered in human non-small cell lung cancers (NSCLC) compared to normal lung tissue. To address this, Northern blot analyses were performed using c-fos, c-jun and EGR1 probes on RNA extracted from 101 NSCLC tumor specimens and adjacent uninvolved lung tissue. Analysis of this cohort revealed that 72% of the normal tissues demonstrate significantly greater expression of these transcription factors as compared to adjacent malignant tissue. Moreover, this expression pattern appeared to be coordinate for all three genes in the majority of cases. This differential expression pattern was confirmed at the protein level using an immunohistochemical approach with antibodies directed against the c-jun, c-fos and EGR1 gene products. Southern blot analyses demonstrated no gross alterations of these sequences at the DNA level, indicating that the observed differential expression pattern was not due to gross structural changes in the genes. These data suggest that down-regulation of these genes may be involved in the pathogenesis of lung cancer.

Tumor suppressor and immediate early transcription factor genes in non-small cell lung cancer.

Non-small lung cancer (NSCLC) is a disease that exhibits multiple genetic lesions. Lung Cancer Study Group (LCSG) 871 was designed to analyze this group of malignancies for alterations in growth factors and/or their receptors, oncogenes, tumor suppressor genes, and immediate early transcription factor genes. Immunohistochemical analysis showed that 32% of evaluable cases studied contained absent or abnormal Rb expression. Sequence analysis of the p53 gene revealed that 58% of these cancers contained structural alterations of this gene, whereas only 45% of these cases overexpressed p53 by immunohistochemical analysis. Finally, both Northern blot and immunohistochemical analysis showed that these tumors exhibited changes in the mRNA and protein expression levels respectively of the immediate early transcription factor genes c-fos, c-jun, and EGR, in that less expression of these genes was evident in the tumors compared with adjacent normal tissue. Understanding both the biologic and molecular significance of these findings may allow us to explore novel modalities for treatment of this disease.

Inactivation of the retinoblastoma susceptibility gene in non-small-cell lung cancer. The Lung Cancer Study Group.

Mutations that prevent the normal expression of the retinoblastoma susceptibility gene (RB) have been linked to the pathogenesis of several human malignancies. Mutational inactivation of this tumor-suppressor gene appears to initiate the development of retinoblastoma, and may also contribute to the pathogenesis of osteosarcomas, soft-tissue sarcomas, small-cell lung cancer and other malignancies. In cooperation with the Lung Cancer Study Group, we studied the structure and expression of the RB gene in a cohort of 219 primary non-small-cell lung cancers (NSCLCs). RB gene structure was studied at the DNA level by Southern blot and chromosome 13 restriction fragment length polymorphism analyses. Expression of the RB gene was evaluated by Northern analysis of the transcript and immunohistochemical analysis of the protein (p105RB). Immunohistochemistry of the RB protein proved to be the most sensitive method for the detection of Rb gene inactivation. Absent or abnormal RB protein staining was detected in 53/163 (32%) evaluable cases studied, while Northern analysis showed 22/219 cases to have an altered or absent RB transcript. Southern analysis revealed only two cases of structural alteration of the gene, but loss of heterozygosity from chromosome 13 was common in tumors that failed to express the protein. Analysis of the clinical outcomes of the patients whose tumors were studied did not show any correlation of RB inactivation with time to relapse or death. The data from this study indicate that the RB gene is inactivated in a significant number of NSCLCs. The role that these mutations may play in the development of NSCLC remains to be defined.

Studies of the retinoblastoma gene in human sarcomas.

The retinoblastoma susceptibility gene, (RB), is a tumor suppressor gene which, when deleted is associated with the development of retinoblastoma. The observation that children with heritable retinoblastoma frequently develop second malignancies, principally sarcomas, led to the detection of similar RB gene deletions in some osteosarcomas. We studied 44 unselected sarcomas from patients with no antecedent retinoblastoma to determine the prevalence and nature of RB gene alterations. DNA and RNA were extracted from fresh tumors and analysed by Southern and Northern blotting. Three of nine osteosarcomas and 4 of 29 soft-tissue sarcomas had deletions of the RB gene. Four of these were full-length, and three were partial deletions of the gene. RNA from 5 of the 7 deleted tumors was studied, and 4 cases completely lacked the RB transcript. Transcripts were found in 19 of 20 tumors with an apparently intact RB gene, and in all the normal tissues studied. An additional tumor lacked the RB transcript, but the gene appeared intact at the DNA level. In total, 8 of 38 sarcomas were found to have alterations of the RB gene. These data indicate that the RB gene is inactivated in a significant number of sarcomas unrelated to retinoblastoma, and that the potential role for the gene in the pathogenesis of human malignancy may not be limited to retinoblastoma.