Human small-cell lung cancers show amplification and expression of the N-myc gene

Abstract

We have found that 6 of 31 independently derived human small-cell lung cancer (SCLC) cell lines have 5-to 170-fold amplified N-myc gene sequences, The amplification is seen with probes from two separate exons of N-myc, which are homologous to either the second or the third exon of the c-myc gene. Amplified N-myc sequences were found in a tumor cell line started prior to chemotherapy, in SCLC tumor samples harvested directly from tumor metastases at autopsy, and from a resected primary lung cancer. Several N-myc-amplified tumor cell lines also exhibited N-myc hybridizing fragments not in the germ-line position. In one patient's tumor, an additional amplified N-myc DNA fragment was observed and this fragment was heterogenously distributed in liver metastases. In contrast to SCLC with neuroendocrine properties, no nonsmall-cell lung cancer lines examined were found to have N-myc amplification. Fragents encoding two N-myc exons also detect increased amounts of a 3.1-kilobase N-myc mRNA in N-mycamplified SCLC lines and in one cell line that does not show N-myc gene amplification. Both DNA and RNA hybridization experiments show that in any one SCLC cell line, only one mycwrelated gene is amplified and expressed. We conclude that N-myc amplification is both common and potentially significant in the tumorigenesis or tumor progression of SCLC.Gene amplification represents an important adaptive mechanism allowing selective increased expression of genes whose products are needed by the cell (1, 2). Thus, the finding of amplification and expression of protooncogenes in tumor cells suggests an important role for the protooncogene product in the cellular and clinical biology of these tumors (3-9). We reported previously that eight independent human small-cell lung cancer (SCLC) lines have stable amplification and increased expression or deregulated expression alone of the c-myc gene (6, 10). Recent studies have shown increased expression with or without amplification of a c-myc-related gene, N-myc, in tumors of neural origin, including neuroblastoma and retinoblastoma (7)(8)(9). In this paper, we show that six independently derived SCLC lines that express neuroendocrine properties (10-12) also have amplified Nmyc sequences and increased levels of N-myc expression compared to non-N-myc-amplified cell lines. In addition, tumor tissues harvested directly from three of these SCLC patients, including one from an untreated primary tumor, also show amplification of the N-myc gene. These results demonstrate that either c-myc or N-myc has been found amplified in 14 of 31 different SCLC lines. This suggests that expression with or without amplification of a myc-related gene may play a prominent role in the biology of SCLC. MATERIALS AND METHODSCloned DNA Fragments. Specific cloned DNA fragments purified from vector sequences were used as probes and are described in the various figure legends. The human c-myc plasmids were a gift of Philip Leder and were both obtained from a 12.7-kilobase (kb) EcoRI fragment isolated and cloned ...