A domain of DNA designated N-myc is amplified 20- to 140-fold in human neuroblastoma cell lines but not in cell lines from other tumor types. N-myc has now been found to be amplified in neuroblastoma tissue from 24 of 63 untreated patients (38 percent). The extent of amplification appears to be bimodal, with amplification of 100- to 300-fold in 12 cases and 3- to 10-fold in 10 others. Amplification was found in 0 of 15 patients with stage 1 or 2 disease, whereas 24 of 48 cases (50 percent) with stage 3 or 4 had evidence of N-myc amplification. These data indicate that N-myc amplification is a common event in untreated human neuroblastomas. Furthermore, N-myc amplification is highly correlated with advanced stages of disease (P less than 0.001) and with the ability to grow in vitro as an established cell line, both of which are associated with a poor prognosis.

https://science.sciencemag.org/content/sci/224/4653/1121.full.pdf

Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage

Eighty-nine patients with untreated primary neuroblastomas were studied to determine the relation between the number of copies of the N-myc oncogene and survival without disease progression. Genomic amplification (3 to 300 copies) of N-myc was detected in 2 of 16 tumors in Stage II, 13 of 20 in Stage III, and 19 of 40 in Stage IV; in contrast, 8 Stage I and 5 Stage IV-S tumors all had 1 copy of the gene (P less than 0.01). Analysis of progression-free survival in all patients revealed that amplification of N-myc was associated with the worst prognosis (P less than 0.0001); the estimated progression-free survival at 18 months was 70 per cent, 30 per cent, and 5 per cent for patients whose tumors had 1, 3 to 10, or more than 10 N-myc copies, respectively. Of 16 Stage II tumors, 2 with amplification metastasized, whereas only 1 of 14 without amplification did so (P = 0.03). Stage IV tumors with amplification progressed most rapidly: nine months after diagnosis the estimated progression-free survival was 61 per cent, 47 per cent, and 0 per cent in patients whose tumors had 1, 3 to 10, or more than 10 copies, respectively (P less than 0.0001). These results suggest that genomic amplification of N-myc may have a key role in determining the aggressiveness of neuroblastomas.

Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas.

DEFINITIONS OF CELLULAR ONCOGENES 555 c-one's: The Proto-Oncogenic Progenitors of Retroviral Oncogenes . . . . . . . . . . . . . . . . . . . 555 Identification of Cellular Oncogenes by Functional Tests for Dominant Mutations 558 Using Genetic Rearrangements to Identify Oncogenes . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562 Proviral insertions can activate expression oj cellular proto-oncogenes 563 Oncogenes at translocation breakpoints. . ... . . . . ..... 566 Oncogenes in amplified DNA ..... . . ...... ........ . . . ..... ........ . . . .. . . ....... ... . . . . . .. . 567 MOLECULAR PROFILES OF ACTIVATED ONCOGENES 570 Base Substitutions in Mutant ras Alleles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 Insertionally Mutated c-myc Loci. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574 Locations oj proviruses 574 Additional mutations in proviraUy mutated loci ...... . . .... 576 Characteristics of Other Proviral Insertion Mutations in Proto-Oncogenes 578 Structural and Functional Properties of Translocation Breakpoints near Cellular Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581 General Jeatures oj c-myc; immunoglobulin gene translocations . . . . . . . . . . . . . . . . . . . . 581 Specific examples of c-myc; Ig translocations in PC and BL lines ........ . . . . . . . . . 583 The Phi chromosome .. . .. ....... . . . . . . . ..... . .. . ... . . . . . . . . ... . . . . . . . ... .. .. .. 586 Other translocations and rearrangements in B cell tumor lines . . . . . . . . . . . . . . . . . . . . . 587 Structural Features oj Amplified Oncogenes . ...... ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587 FROM c-onc TO v-onc: COLLUSION OF MUTATIONAL MECHANISMS....... 588 The Favored Model Jar Transduction of Oncogenes . .. . . 590 The Functional Significance of Diff erences Between c-onc' s and v-onc' s 592 INACTIVATION OF INTEGRATED v-onc GENES: A SAMPLER OF MUTATIONAL MECHANISMS 593

The Molecular Genetics of Cellular Oncogenes

Publisher Summary This chapter summarizes the third mechanism of oncogene activation—oncogene amplification. It illustrates the various ways by which the oncogenic potential of different proto-oncogenes can be activated. Because of the involvement of myc oncogenes in amplifications in a variety of tumors, other lesions that also activate the cellular oncogene c-myc and the aspects of the normal regulation of this oncogene are also described. Since its discovery in drug-resistant eukaryotic cells, somatic amplification of specific genes has been implicated in an increasing variety of adaptive responses of cells to environmental stresses. Experimental work on drug-resistant cells has shown that in the absence of a selection pressure (drug), double minute chromosomes (DMINs) and the amplified genes that they contain are lost, whereas amplified DNA in the form of homogeneously staining regions (HSRs) is retained in the cells. If DMINs and HSRs contain amplified genes that encode drug-resistant or growth-stimulating protein products, it would follow that the more stable chromosomal form, the HSR, confers a greater selective growth advantage for cells. Although DMINs and HSRs have been described predominantly in tumor cells selected for the resistance to cytotoxic drugs, it is also clear that DMINs and HSRs may be present in cancer cells before the start of therapy.

Oncogene amplification in tumor cells

The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc.

The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression.

Several unusual chromosome structures have been described in drug-resistant cell lines and in certain tumours. These structures include elongated homogeneously staining regions (HSRs), small extrachromosomal paired chromatin bodies (double minutes, DMs) and abnormally banded regions (ABRs) with strong but anomalous band patterns1–3. There is evidence that these are alternative forms of gene amplification, with HSRs breaking down to form DMs, and DMs integrating into the chromosome to generate HSRs and ABRs5–7. Recently, it was demonstrated that, compared with several normal leukaemia human cells, DNA sequences representing the human homologue of the onc gene of the avian myelocytomatosis virus (MC29), the so-called c-myc gene, were amplified in HL-60 cells11,12. This is a human pro-myelocytic leukaemia cell line established in the laboratory of one of us (R.C.G.) at the National Cancer Institute (Bethesda, Maryland) in 1977, and widely used for studies on myeloid and monocytic differentiation8–10. Amplification of the gene was present in primary leukaemic cells of the patient12, and DMs were noted in some of these cells as well as in early passages of the HL-60 line13. No structure resembling HSRs or ABRs were noted in karyotypic studies at this early stage13 and there were no alterations involving the long arm of chromosome 8 (8q), to which the c-myc gene has recently been mapped14–16. We have now re-examined the karyotype of the HL-60 line, using cells frozen at various times during its continuous passage at the Wistar Institute (Philadelphia, Pennysylvania) to look for chromosomal abnormalities that might be associated with the amplification of c-myc. We find that, beginning in 1979, HL-60 cells at the Wistar Institute no longer had DMs, but did show an abnormal 8q+ chromosome, replacing a normal chromosome 8, and representing an ABR reflecting the site of myc gene amplification.

Romanczuk H

Papers

Association of amplified oncogene c- myc with an abnormally banded chromosome 8 in a human leukaemia cell line