Transformation of rodent immortalized and embryo cells by oncogenes. I. Mouse (NIH 3T3) and rat (FR 3T3) immortalized fibroblasts

Immortalized mouse NIH 3T3 cells were transformed by gene transfer of DNA isolated from a human bladder tumor cell line and plasmids containing an activated human Ha-ras oncogene insert. For gene transfer the calcium-phosphate co-precipitation method was used. Transformation was evaluated by morphological focus formation, growth in soft agar and tumor development in nude mice. In addition, immortalized rat FR 3T3 cells were transformed by Ha-ras, too. The co-transfer of ras and myc oncogenes did not enhance focus formation in FR 3T3 cells.     

Resistance of human cells to tumorigenesis induced by cloned transforming genes.

The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse. The EJ gene, a mutant form of the cellular c-Ha-ras gene, actively transforms NIH/3T3 mouse cells and CHEF/18 hamster cells but is inactive in FS-2 cells. Morphological transformation, focus formation, and tumorigenicity in nude mice were not induced when EJ DNA was transfected into FS-2 cells by using the selectable vector pSVgptEJ. The intactness of the transfected EJ DNA was established by restriction fragment analysis. This result raises the question of what role, if any, the mutated gene derived from the EJ cells played in the origin of the EJ bladder carcinoma.     

Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Ha-ras-1 gene derived from the human T24 bladder carcinoma cell line and compared them with untransfected NIH/3T3 cells. Whereas subcutaneous implantation of NIH/3T3 cells in the supraclavicular region produced palpable tumors that failed to metastasize, NIH/3T3 cells inoculated in the footpad gave rise to malignant tumors that metastasized to the lung. Under identical conditions and irrespective of the site of implantation, A51 cells formed rapidly growing primary tumors that produced pulmonary metastases. In an assay for experimental metastasis, intravenously injected NIH/3T3 cells gave rise to pulmonary nodules only at high cell inocula and in long-term survivors (90 days after injection). In contrast, A51 cells formed multiple lung tumor colonies detectable 14 days after injection. These results indicate that "normal" untransfected NIH/3T3 cultures contain subpopulations of cells that express malignant properties and that transfection of NIH/3T3 cells with activated c-Ha-ras-1 accelerates formation of metastases.     

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: sequence analysis of an enzymatically amplified mutant HRAS allele.

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies (11 X 10(-4) consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[a]pyrene (+/-)anti- 7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed i in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. Direct sequence analysis of the amplified DNA indicated equal presence of a wild-type (GGC) and mutant (GTC) allele of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC----TA codon 12 transversions that have been commonly observed in human tumors. This oncogene as well as yet to be identified oncogene are also shown to stably confer anchorage-independence to human cells.     

Amplification and rearrangement of the Kirsten ras oncogene in virus-transformed BALB/c 3T3 cells during malignant tumor progression.

Analyses of the cellular and viral Kirsten ras genes (c-Ki-ras and v-Ki-ras, respectively) during malignant tumor progression were performed by using Kirsten murine sarcoma virus-transformed BALB/c 3T3 cells that harbor a replication-defective provirus. After injection into athymic nude mice by four different routes, primary tumors and secondary lung metastases were isolated, adapted to in vitro growth, and analyzed for DNA levels and mRNA expression of both genes for comparison with the originally injected transformed cells and untransformed 3T3 cells. For all tumors (primary or secondary), the v-Ki-ras gene was amplified and v-Ki-ras mRNA expression was highly elevated above that observed in the original transformed cell population. In two of five lung metastases from the i.v. and footpad injection routes, rearranged Ki-ras DNA sequences were observed. Micrometastases from the s.c. route of injection did not display these alterations. Injection of footpad lung tumor cells with rearrangements into a second group of animals led to multiple lung metastases with even further rearrangements correlating with more effective lung colonization/growth ability (overt lung tumors in five of eight animals less than 20 days after injection). However, reinjection of an i.v. lung tumor with rearranged Ki-ras led to no further rearrangements in the lung microfoci tumors isolated greater than 40 days after injection. These data suggest (i) the significance of amplification and elevated expression of v-Ki-ras in tumor formation, (ii) correlation of this amplification with more effective tumor progression, and (iii) the selective advantage that cells with Ki-ras DNA sequence additions have in the formation of overt lung tumors.     

IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein.

The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. (i) Extracts of yeast cells overexpressing IRA2 stimulated the GTPase activity of the yeast RAS2 protein. (ii) An epitope for a monoclonal antibody (12CA5) was added to the N terminus of the IRA2 protein. The GAP activity of extracts prepared from cells expressing this fusion protein was shown to be immunoprecipitable by 12CA5. (iii) An IRA2 protein fused to glutathione S-transferase (GST) was produced and partially purified from Escherichia coli cells. GAP activity was detected with this purified GST-IRA2 fusion protein. (iv) The GAP activity of IRA2 proteins described above did not stimulate the GTPase activity of the RAS2Val19 protein, a protein having an amino acid alteration analogous to that found in mammalian oncogenic ras proteins. This result parallels studies showing that mammalian GAP is incapable of stimulating the GTPase activity of mammalian oncogenic proteins. The remarkable conservation between the GAP activity in mammalian and yeast cells supports the idea that the function of GAP is to negatively regulate ras proteins in mammalian cells.     

A product of yeast RAS2 gene is a guanine nucleotide binding protein.

Yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, which show remarkable homology to mammalian ras genes. To characterize these gene products, we have expressed the RAS2 gene in yeast using an inducible GAL10 promoter. After labeling with [35S]methionine and immunoprecipitating with a monoclonal antibody Y13-259, which reacts with p21 encoded by mammalian ras genes, a major band having an apparent molecular weight of 41,000 is detected. This band has also been identified in cell-free translation products of polyadenylated RNA extracted from yeast cells grown in the presence of galactose. Crude extracts of cells expressing the RAS2 gene exhibit guanine nucleotide binding activity. This is detected by incubation with [3H]GDP followed by immunoprecipitation with the antibody Y13-259. The binding of labeled GDP is inhibited by a 20-fold excess of GDP, GTP, and, to a lesser extent, by UTP, a characteristic similar to that possessed by the mammalian ras proteins. However, the activity of the yeast protein differs from that of the mammalian proteins in its strong dependence on temperature. The guanine nucleotide binding activity provides an assay to purify the yeast protein.     

Tumourigenicity of MTG8, a leukaemia-related gene, in concert with v-Ha-ras gene in BALB/3T3 cells

The MTG8 ( ETO ) gene has been identified as the translocation partner of AML1 ( PEBP2αB or CBFα2 ) gene in the AML1 / MTG8 ( ETO ) fused gene caused by t(8;21) translocation in human acute myeloid leukaemia, M2 type. Although AML1/MTG8 chimaeric protein is known to inhibit the functioning of AML1 protein, the precise function of MTG8 gene itself is not known yet. We studied the significance of MTG8 gene in the oncogenicity of AML1 / MTG8 fused gene, by introducing full-length MTG8 cDNA into both BALB/3T3 cells containing v-Ha- ras gene (Bhas 42 cells) and BALB/3T3 cells without v-Ha- ras gene.
Irrespective of the overexpression of MTG8 gene in both groups of cells, Bhas- MTG8 clones which contained v-Ha- ras gene and expressed the MTG8 gene at a level more than twice that of parental Bhas 42 cells induced cell transformation, whereas BALB- MTG8 clones without v-Ha- ras gene did not. Furthermore, injection of the transformed Bhas- MTG8 clones into the subcutaneous tissue of nude mice induced tumours, whereas that of BALB- MTG8 clones did not. These results suggest that MTG8 gene product, in cooperation with viral Ras protein, resulted in tumour formation. We provide the first evidence that MTG8 gene by itself has a carcinogenic property within the AML1 / MTG8 ( ETO ) fused gene.     

Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice.

Ras oncogenes owe their transforming properties to single point mutations in the sequence coding for the active site of the p21 protein. These mutations lead to changes in cellular proliferation and induce tumorigenic properties. Point mutations represent a well-defined target for antisense oligonucleotides that can specifically suppress the translation of the targeted mutant mRNA. We show that the stability and cellular disponibility of antisense oligonucleotides can be markedly improved by adsorption to polyalkylcyanoacrylate nanoparticles. Nanoparticle-adsorbed antisense oligonucleotides directed to a point mutation (G-->U) in codon 12 of the Ha-ras mRNA selectively inhibited the proliferation of cells expressing the point-mutated Ha-ras gene at a concentration 100 times lower than free oligonucleotides. In addition they markedly inhibited Ha-ras-dependent tumor growth in nude mice after subcutaneous injection. These experiments show that inhibition of ras oncogenes by antisense oligonucleotides can block tumor development even though ras oncogenic activation might be an early event in tumor progression.     

A mouse model for EML4-ALK-positive lung cancer

EML4-ALK is a fusion-type protein tyrosine kinase that is generated in human non-small-cell lung cancer (NSCLC) as a result of a recurrent chromosome inversion, inv (2)(p21p23). Although mouse 3T3 fibroblasts expressing human EML4-ALK form transformed foci in culture and s.c. tumors in nude mice, it has remained unclear whether this fusion protein plays an essential role in the carcinogenesis of NSCLC. To address this issue, we have now established transgenic mouse lines that express EML4-ALK specifically in lung alveolar epithelial cells. All of the transgenic mice examined developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth, confirming the potent oncogenic activity of the fusion kinase. Although such tumors underwent progressive enlargement in control animals, oral administration of a small-molecule inhibitor of the kinase activity of ALK resulted in their rapid disappearance. Similarly, whereas i.v. injection of 3T3 cells expressing EML4-ALK induced lethal respiratory failure in recipient nude mice, administration of the ALK inhibitor effectively cleared the tumor burden and improved the survival of such animals. These data together reinforce the pivotal role of EML4-ALK in the pathogenesis of NSCLC in humans, and they provide experimental support for the treatment of this intractable cancer with ALK inhibitors.     

Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.

The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.     

Transforming genes of human hematopoietic tumors: frequent detection of ras-related oncogenes whose activation appears to be independent of tumor phenotype.

We surveyed 22 human hematopoietic tumors and tumor cell lines for sequences capable of transforming NIH 3T3 cells by DNA transfection. A primary human acute myelogenous leukemia, a chronic myelogenous leukemia cell line, and cell lines derived from three independent acute lymphocytic leukemias demonstrated oncogenes capable of conferring the transformed phenotype to NIH 3T3 cells through serial cycles of transfection. One of three transforming genes associated with acute lymphocytic leukemia cells (classified as thymocyte developmental stage II) was identified as the activated cellular homologue of the Kirsten murine sarcoma virus onc gene, kis, a member of the ras family of onc genes. A transforming gene, which was demonstrated to be common to several human myeloid and lymphoid tumor cells, was shown to be a distantly related member of the ras gene family. Thus, the NIH 3T3 transfection assay commonly detects related oncogenes in human hematopoietic tumor cells. Moreover, the activation of these oncogenes appears to be independent of the specific stage of cell differentiation or tumor phenotype.     

Isolation of genes specifically expressed in flat revertant cells derived from activated ras-transformed NIH 3T3 cells by treatment with azatyrosine.

We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21ras is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the ras-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), which was identified as a tumor-suppressor gene by Contente et al. [Contente, S., Kenyon, K., Rimoldi, D. & Friedman, R. M. (1990) Science 249, 796-798]. Other genes identified were the collagen type III and rhoB genes. Approximately half the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including rrg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype.     

Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses.

Blot hybridization analysis indicated that NIH 3T3 mouse bladder transformed by high molecular weight DNAs of a human bladder and a human lung carcinoma cell line contained new sequences homologous, respectively, to the transforming genes of Harvey (rasH) and Kirsten (rasK) sarcoma viruses. The unique ras sequences were present in multiple independent NIH cell lines transformed in both primary and secondary transfection assays and corresponded to ras sequences normally present in human DNAs. The ras gene product was expressed in NIH cells transformed by bladder carcinoma DNAs and in the human bladder carcinoma cell lines at levels 2- to 4-fold greater than the level observed in nontransformed NIH 3T3 cells. These results indicate that the transforming genes of these human tumor cell lines are the cellular homologs of two retroviral transforming genes.     

The human Ha-ras oncogene induces genomic instability in murine fibroblasts within one cell cycle.

Many human tumors contain an activating mutation in one of the ras protooncogenes. Additionally, these tumor cells are often heteroploid and characterized by chromosome breaks and rearrangements that are consequences of the genomic instability that is thought to contribute to tumor progression. The concurrence of ras mutations and genomic instability in tumors prompted us to ask whether selective induction of an activated Ha-ras gene could render a genome unstable. The NIH 3T3 cells used in this study contained mutant p53 genes and carried a selectively inducible activated (EJ) Ha-ras transgene under the control of bacterial lactose regulatory elements. When stably transfected cells were induced to express activated Ha-ras by isopropyl beta-D-thiogalactoside administration, there was a marked increase in the number of gross chromosomal aberrations including acentric fragments, multicentric chromosomes, and double minutes, which occurred within the time frame of a single cell cycle from the time of induction. To confirm that these aberrations occurred within the first cell cycle after mutant Ha-ras induction, the cells were arrested in G1 phase by serum depletion and, subsequently, released by administration of isopropyl beta-D-thiogalactoside or serum. The mitoses from cells released with isopropyl beta-D-thiogalactoside contained a 3-fold elevation in the fraction of chromosomes containing aberrations compared to mitoses from parallel cell cultures that were released with serum. Thus, the induction of activated Ha-ras gene expression in these cells results in genomic instability that can be detected as aberrant chromosomes at the next mitosis.     

Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line.

DNA from the human neuroblastoma cell line SK-N-SH is capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. Using genetic selection with the Escherichia coli sup F gene, we have isolated human sequences from mouse cells responsible for the oncogenic transformation. These sequences are present in all human DNAs surveyed and no gross rearrangements of these sequences are found in SK-N-SH cells. Although clearly distinct from two other human transforming genes present in bladder, lung, and colon carcinoma cell lines, all three transforming gene sequences may be related members of the ras gene family.     

Novel experimental models of human cancer metastasis in nude mice: Lung metastasis,intraabdominal carcinomatosis with ascites,and liver metastasis

Summary The ability of RPMI4788 cells, a human colon cancer cell line, to produce experimental metastases in the lung, intraperitoneal cavity, and liver was studied in nude mice. Injection of 2×10⁶ tumor cells into the tail vein of nude mice produced metastatic lung tumors, and an intraportal injection of 5×10⁶ cells produced metastatic liver tumors. An intraabdominal carcinomatosis with ascites was formed after an i.p. injection of 5×10⁶ tumor cells. The nude mice with lung metastasis or intraabdominal carcinomatosis always died within a few weeks. Macroscopic observation showed that the number of lung metastatic nodules on day 21 after tumor inoculation was 311.3±78.2 (mean±SD) in BALB/C nude mice, and 187.5±26.7 in ICR nude mice. In survival experiments, the mice with intraabdominal carcinomatosis showed a mean survival of 29.0±1.7 (mean±SD) days in BALB/C nude mice and 43.6±6.1 days in ICR nude mice. These novel experimental models of metastases in nude mice produced by injection of RPMI4788 cells had high reproducibility and may be useful not only for the study of the metastatic process but also for testing anticancer drugs.     

Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA.

NIH 3T3 mouse fibroblasts form nonmetastasizing fibrosarcomas upon transformation by the Ha-ras oncogene isolated from the EJ human bladder carcinoma cell line and subcutaneous inoculation into immunocompetent NFS/NCr mice. DNA from a human metastatic tumor was transfected into these Ha-ras transformants, and one of the resulting colonies yielded a lung metastasis after subcutaneous inoculation. DNA was isolated from this metastasis and subjected to a second round of transfer into Ha-ras-transformed NIH 3T3 cells. Inoculation of these transfected cultures into mice led once again to formation of metastases, this time at a higher frequency. Examination of four of the resulting metastases revealed discrete human DNA fragments that were common to all four. These findings demonstrate that the metastatic phenotype can be transferred via DNA from cell to cell and is associated with the presence of a discrete DNA segment. This segment is not identical to the myc oncogene or to any of the frequently detected ras tumor oncogenes.     

Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse.

Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo.     

Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation.

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.