Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as doubleminute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.

Oncogene amplification in tumor cells results in the overexpression of proteins that confer a growth advantage in

Amplification of oncogenes has been found to be an important prognostic factor in behavior of patients' malignancies. In this study we have used new gel electrophoresis techniques to follow the location of amplified c-myc oncogene sequences in HL-60 promyelocytic leukemia cells. In passages 46-62 of the cells, the cells contain amplified c-myc sequences on submicroscopic circular extrachromosomal DNA (episomes). With increased passages in culture (passages 63-72) the cells lose the episome c-myc sequences with a shift of those sequences to double minutes. With additional passage in culture, the c-myc shifts from the double minutes to a chromosomal site der(5)t(5; 17Xqll.2;q?11.2). Concomitant with the shift of the c-myc sequences into the chromosomal compartment is a phenotypic change of a shortened cell-doubling time. These studies provide the first molecular evidence of a progression from a submicroscopic location for amplified oncogene sequences to a chromosomal location for the amplified sequences. This molecularly documented model can now be used to test various strategies to prevent incorporation of extrachromosomally located oncogene sequences into chromosomal sites. Prevention of integration of the oncogene sequences into chromosomal sites could modulate progression of patients'

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A human tumor cloning assay was used to analyze drug sensitivity profiles for 99 pairs of tumor samples taken simultaneously from the same patient. These pairs included two areas of the same primary (12 pairs), primary tumor versus metastasis (29 pairs), and metastasis versus metastasis (45 pairs). In addition, to assess variability in the culture and sensitivity techniques, 13 specimens were divided equally and processed twice. One hundred fourteen evaluable drug sensitivity tests were performed on the specimen pairs. Drug sensitivity profiles for the same specimen processed twice demonstrated good agreement (P = 0.03). Sensitivity profiles from two different areas of the same primary were also quite similar (P = 0.002). However, when one sampled a primary tumor and its metastasis for drug sensitivity testing, the agreement (in terms of percent survival) was poor (P = 0.84). Agreement was also poor for drug sensitivity profiles of one metastasis versus another metastasis (P = 0.39). This heterogeneity in drug sensitivity profiles of primary versus metastasis and of metastasis versus metastasis could account for some of the false positives and false negatives noted in clinical correlation trials with the cloning assay. It may also have implications for adjuvant treatment programs where chemosensitivity of the primary tumor can be determined, but will probably not be predictive for chemosensitivity of the micrometastases.