The presence and synthesis of c‐myc protein and mRNA in the cell cycle has been studied. We find that c‐myc mRNA is present, at equivalent levels, at all times in the cell cycle with the possible exception of mitosis. Furthermore, we demonstrate that this mRNA is transcribed in both G1 and G2 phases. An analysis of the c‐myc protein in vivo shows that de novo synthesis occurs in G1 and G2 and the protein turns over with a half‐life of approximately 20‐30 min in both phases. Furthermore, the level of c‐myc protein rapidly increases in cell populations when they re‐initiate the cell cycle, thereafter decreasing as the culture reaches quiescence. The results therefore suggest that expression of c‐myc can be rapidly modulated and that it is activated during the G0 to G1 transition, but is expressed thereafter in the cell cycle.

The human c-myc gene consists of three exons transcribed from two distinct promoters and the function of the first, noncoding exon is unknown. In COLO 320 cells, there co-exist normal and truncated (i.e., lacking exon 1) c-myc genes, both of which are transcribed. Studies on the turnover of c-myc mRNA show that the normal mRNA has an in vivo half-life of -30 min which is approximately similar to the turnover time of the mRNA in lymphoblastoid cells. However, the truncated mRNA was found to be substantially more stable. This observation was also made with a Burkitt's lymphoma cell line which has a translocated, truncated c-myc gene. Therefore truncation of the c-myc gene can cause the mRNA to be more stable than the full size product suggesting that this can be a crucial factor in the activation of the c-myc oncogene, by exon 1 loss, in chromosomal translocation. The results also suggest a role for exon 1 in the c-myc mRNA degradative mechanism.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

Summary Amplification and expression of the mdrl gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdrl gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.Resistance of tumours to multiple drugs is a major problem in cancer treatment. Studies using in vitro derived multidrugresistant (MDR) cell lines have shown that MDR is often associated with over-production of two groups of proteins: the P-glycoproteins (for review see which have drug-binding properties (Safa et al., 1986) al., 1982;Gros et al., 1986;Kartner et al., 1983;Robertson et al., 1984;Scotto et al., 1986;Shen et al., 1986b;Van der Bliek et al., 1986b).In an effort to elucidate mechanisms of MDR in human small cell lung cancer (SCLC), multidrug resistant variants (MDR) of human SCLC cell line NCI-H69, have recently been derived following cell culture in increasing doses of adriamycin (ADM) Mirksi et al., 1987). Surprisingly, the MDR variant H69/AR (Mirski et al., 1987) does not show amplification, rearrangement or overexpression of the P-glycoprotein gene (Trent et al., 1988) suggesting that other factors are responsible for the MDR phenotype in these cells. The present study investigates Pglycoprotein gene amplification and expression in H69/LX4 (Twentyman et al., 1986) a second, independently derived MDR variant of NCI-H69. We report that, in marked contrast to H69/AR cells, amplification and hyperexpression of P-glycoprotein gene occurs in this MDR cell line. Materials and methods Cell linesThe SCLC cell line NCI-H69 (kindly supplied by Drs Desmond Carney and Adi Gazdar of the NCI Navy Medical Oncology Branch, Bethesda, MD) was derived from a patient who had previously received multidrug therapy (including ADM).Full details of the in vitro derivation of the MDR variant of NCI-H69 are given elsewhere . Briefly, NCI-H69 parent (H69P) cells were initially exposed to 0.02 pgml-1 ADM and then transferred to 0.04pgml-1 ADM after 3 weeks. After a further 4 weeks, ADM was removed and when cell growth resumed, ADM was reintroduced at weekly increasing doses of 0.1, 0.2 and