ras oncogenes and phenotypic staging in N-methylnitrosourea- and gamma-irradiation-induced thymic lymphomas in C57BL/6J mice

We have investigated stages of thymic lymphoma development in radiation and N-methylnitrosourea (NMU)-treated C57BL/6J mice. The lymphoma cell was identified serologically as a cortical population bearing MEL-14hi, H-2Khi, and IL-2R+ surface markers. According to these parameters in C57BL/6J mice the lymphoma cell was the same regardless of inducing agent or activated oncogene (ras or non-ras). Transforming activity in the radiation and NMU-induced tumors was analyzed using both the nude mouse tumorigenicity assay and the focus-forming assay. 8/10 NMU-induced tumors and 12/15 radiation-induced tumors showed transforming activity in the tumorigenicity assay. Southern blot analysis of the nude mouse transformants demonstrated K-ras transforming sequences in eight of eight NMU-induced lymphoma DNAs, two of 12 radiation-induced lymphoma DNAs and N-ras transforming sequences in five of 12 radiation-induced lymphoma DNAs. The non-ras transforming activity in five DNAs from radiation-induced thymic lymphomas indicates the presence of an unidentified oncogene(s) in these tumors. Staging of thymic lymphoma development in this animal model system will allow the study of oncogene activation early in the course of carcinogen-induced disease. These results also emphasize the high sensitivity of the nude mouse assay to score for activated oncogenes and might also indicate a high frequency of K-ras activation in NMU-induced lymphomas in C57BL/6J mice.     

Detection of transforming oncogenes in rat colon tumors induced by direct perfusion with N-methyl-N-nitrosourea.

We assayed rat colon tumors induced by N-methyl-N-nitrosourea (MNU) for transforming oncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA from 3 of 3 adenomas and 3 of 5 carcinomas induced transformed foci on NIH 3T3 cells. DNA from 2 of 3 primary foci also possessed focus-forming activity, and rat-specific sequences were observed in secondary focus DNAs. Furthermore, NIH 3T3 cells transfected with DNA from a carcinoma and from a primary focus derived from it, both positive in the focus-forming assay, induced tumors in nude mice. We found no evidence for rat H-ras, K-ras, or N-ras sequences in the DNA of any of 16 primary foci derived from 6 rat tumors; thus, in contrast to other animal tumor models induced by MNU, activation of the ras genes does not appear to predominantly occur in MNU-induced rat colon tumors. We also did not observe, in any of these foci, sequences corresponding to the rat neu, raf, fms, met, or hst genes, thus indicating that none of these is the transforming oncogene in our model. These results suggest that an as yet unidentified transforming oncogene may be activated in rat colon tumors induced by MNU.     

Activation of protooncogenes in spontaneously occurring non-liver tumors from C57BL/6 x C3H F1 mice

The C57BL/6 x C3H F1 (hereafter called B6C3F1) mouse is an important animal model for long-term carcinogenesis studies. Maintained under normal laboratory conditions, these mice develop various types of spontaneous tumors during their lifetime. Activated Ha-ras genes have been detected in 66% of spontaneous hepatocellular tumors in the B6C3F1 mouse [Reynolds et al., Science (Washington DC), 237:1309, 1988]. In this study 49 spontaneous non-liver tumors were investigated for oncogene activation by DNA transfection techniques. Of the 49 tumor DNAs analyzed, only 5 yielded multiple foci in the NIH 3T3 focus assay: 2 of 10 pulmonary adenocarcinomas; 0 of 25 lymphomas; 2 of 2 Harderian gland adenomas; 0 of 1 adenocarcinoma of the small intestine; 1 of 6 malignant skin tumors; 0 of 4 hemangiosarcomas; and 0 of 1 lung metastasis of a hepatocellular carcinoma. DNA from six lymphomas which were negative in the NIH 3T3 focus assay were further analyzed for transforming genes by the nude mouse tumorigenicity assay. One of the five lymphomas tested positive with this assay. Southern blot analysis identified five activated ras genes: H-ras in two Harderian gland adenomas; K-ras in one pulmonary adenocarcinoma and in one s.c. adenocarcinoma; and N-ras in one lymphoma. The mutations involved were CG to AT and AT to TA in codon 61 of the Ha-ras genes, GC to AT or TA in codon 12 of the K-ras genes, and a GC to AT mutation in codon 12 of the N-ras gene. Transformant DNA from a pulmonary adenocarcinoma which yielded multiple foci in the transfection assay did not hybridize to DNA probes specific for the K-, H-, and N-ras, raf, neu, and met genes. Thirteen additional tumor DNAs yielded a single focus in the NIH 3T3 transfection assay. The transformant DNAs retransmitted in a second cycle transfection assay. Rearranged and/or amplified raf genes were detected in six of the transformant DNAs. At present we do not know whether these activated raf genes were present in the original tumor DNA. The other seven transformant DNAs did not hybridize with any of the above mentioned specific DNA probes utilized in Southern blot analysis. Unlike liver tumors, the activation of ras protooncogenes is not a frequent event in the development of spontaneous non-liver tumors of the B6C3F1 mouse. The results from this study should aid in understanding the neoplastic development associated with exposure to chemical carcinogens in the B6C3F1 mouse.     

Novel transforming sequences in human acute myelocytic leukemia cell lines

DNA transfection analyses using the tumorigenicity assay were performed on seven human acute myelocytic leukemia (AML) cell lines. DNAs from all cell lines induced tumors in nude mice. Respective transforming sequences could be identified as activated N-ras genes in AML cell lines THP-1, KG-1 and Rc2a. We observed the transfer of five putative novel transforming sequences from DNAs of cell lines U937, ML-1, HEL and CTV-2. These sequences appear to be unrelated to each other and previously known oncogenes. Transfections containing these transforming genes show distinctive biological and morphological characteristics. Our data suggest that the transforming sequences reported here became activated during the transfection process rather than the AML cell line.     

Alterations of mouse proto-oncogenes in sarcomas induced after transplantation of human tumors in athymic nude mice

During serial subcutaneous transplantation of several types of human tumors into nude mice, the local development of malignant mouse-specific sarcomas has been observed. Although the frequency of sarcoma induction is low, this phenomenon is very important because the mouse-specific sarcomas completely replaced the human tumors during serial transplantation. The DNA of five independently induced mouse-specific sarcomas was transfected into NIH/3T3 cells in order to detect oncogenes associated with mouse-specific sarcoma induction. Two of these DNAs were found to carry activated mouse c-N-ras and c-Ki-ras genes. The sequence analysis of the molecularly cloned mouse c-N-ras oncogene showed a single nucleotide transition from G to A at the 12th codon. This results in substitution of aspartic acid for glycine at this position. The mouse c-myc gene was also found to be amplified in a sarcoma. In these mouse sarcoma DNAs, human Alu sequences were not detected. These data strongly suggest that the mouse-specific sarcomas were not induced by the transfer of human transforming sequences but by the alterations of mouse proto-oncogenes.     

Alterations of Mouse Proto-oncogenes in Sarcomas Induced after Transplantation of Human Tumors in Athymic Nude Mice

During serial subcutaneous transplantation of several types of human tumors into nude mice, the local development of malignant mouse-specific sarcomas has been observed. Although the frequency of sarcoma induction is low, this phenomenon is very important because the mouse-specific sarcomas completely replaced the human tumors during serial transplantation. The DNA of five independently induced mouse-specific sarcomas was transfected into NIH/3T3 cells in order to detect oncogenes associated with mouse-specific sarcoma induction. Two of these DNAs were found to carry activated mouse c-N- ras and c-Ki- ras genes. The sequence analysis of the molecularly cloned mouse c-N- ras oncogene showed a single nucleotide transition from G to A at the 12th codon. This results in substitution of aspartic acid for glycine at this position. The mouse c- myc gene was also found to be amplified in a sarcoma. In these mouse sarcoma DNAs, human Alu sequences were not detected. These data strongly suggest that the mouse-specific sarcomas were not induced by the transfer of human transforming sequences but by the alterations of mouse proto-oncogenes.     

Detection of distinct transforming genes in X-ray induced tumors

DNAs from mouse skin tumors (papillomas, squamous cell carcinomas,basal cell carcinomas and pilomatrixomas) initiated with X-irradiationand promoted with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)demonstrated dominant transforming activity by the productionof transformed foci in the mouse recipient tine, NIH3T3. Dominanttransforming activity was not found in DNA isolated from normalmouse epidermis or from the corresponding liver. The NIH3T3transformants induced with squamous cell carcinoma DNA grewin soft agar and formed tumors in nude mice. Southern blot analysisof primary NIH3T3 transformant DNAs carrying oncogenes fromradiation-initiated squamous cell carcinomas indicated thatthe oncogenes responsible for the transformation of the recipientcells were not Ha-ras, Ki-ras or N-ras genes, nor were theyerbB, B-lym, met, neu or raf. The data presented indicate thatDNAs from radiation-initiated mouse skin tumors contain dominanttransforming genes that are detectable by DNA-mediated genetransfer. The oncogene sequences activated in these radiation-initiatedtumors are distinct non-ras transforming genes.     

Ras gene mutation and amplification in human nonmelanoma skin cancers

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain activated Ha-ras oncogenes capable of inducing morphologic and tumorigenic transformation of NIH 3T3 cells. In this study, we analyzed human primary squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) occurring on sun-exposed body sites for mutations in codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction, followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to the primary human skin cancers, we also analyzed Ha-ras-positive NIH 3T3 transformants for mutations in the Ha-ras oncogene. The results indicated that all three NIH 3T3 transformants, 11 of 24 (46%) SCCs, and 5 of 16 (31%) BCCs contained mutations at the second position of Ha-ras codon 12 (GGC----GTC), predicting a glycine-to-valine amino acid substitution, whereas only 1 of 40 skin cancers (an SCC) displayed a mutation in the first position of Ki-ras codon 12 (GGT----AGT), predicting a glycine-to-serine amino acid change. In addition, three of the SCCs contained highly amplified copies of the N-ras oncogene in their genomic DNA. Interestingly, two of the SCCs containing amplified N-ras sequences also had G----T mutations in codon 12 of the Ha-ras oncogene. These studies demonstrate that mutations in codon 12 of the Ha-ras oncogene occurred at a high frequency in human skin cancers originating on sun-exposed body sites, whereas mutation in codon 12 of Ki-ras or amplification of N-ras occurred at a low frequency. Since the mutations in the Ha-ras and Ki-ras oncogenes were located opposite potential pyrimidine dimer sites (C-C), it is likely that these mutations were induced by ultraviolet radiation present in sunlight.     

Detection and identification of activated oncogenes in human skin cancers occurring on sun-exposed body sites

High-molecular-weight DNA isolated from eight fresh human skin cancers occurring on sun-exposed body sites were assayed for their ability to transform NIH 3T3 cells. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select cells that had taken up the transfected DNA. About 2 weeks after transfection, G418-resistant colonies were pooled and injected s.c. into athymic nude mice. The NIH 3T3 cells transfected with DNA from six of the human skin cancers induced tumors in nude mice. DNAs from all six tumor cell lines contained human alu sequences. Southern blot hybridization with ras-specific probes revealed that DNAs from the four alu-rich tumors contained the human Ha-ras oncogene, in addition to that of the NIH 3T3 controls. In contrast, DNAs from the other two tumors did not contain any of the known oncogenes tested, except those endogenous to NIH 3T3 cells. DNAs from three of four first cycle tumorigenic transformants gave rise to morphologically transformed foci when assayed in a second cycle of transfection. DNAs from all three secondary transformants contained discrete human alu sequences, and in addition, contained Ha-ras sequences similar to those present in their respective primary transformants. Interestingly, DNA from both primary and secondary transformants of one particular human squamous cell carcinoma contained highly amplified copies of the Ha-ras oncogene. These results suggest that activation of the Ha-ras oncogene may be common in human skin cancers originating on sun-exposed body sites. Further characterization of the Ha-ras oncogenes present in these human skin cancers may provide information on the molecular mechanisms by which UV radiation of the sun induces human neoplasms on exposed body sites.     

Transforming activity of DNA from rat liver tumors induced by the carcinogen methyl(acetoxymethyl)nitrosamine

Altered c-Ha-ras genes have been frequently detected in the DNA of spontaneous or chemically induced mouse liver tumors. To determine if ras gene mutation is a frequent event during liver carcinogenesis in rats, we examined the transforming activity of DNA from liver tumors that developed in rats injected with methyl(acetoxymethyl)nitrosamine (DMN-OAc) after a partial hepatectomy. Three weeks after the injection of DMN-OAc, rats were fed a diet containing phenobarbital. This carcinogen acts only on replicating liver cells. Six of eight tumor DNAs induced the transformation of NIH 3T3 cells. The transforming activity was stable upon a second round of transfection, and the transformants were tumorigenic in nude mice. Southern blot analysis of transformant DNAs showed that the transforming activity was not due to the acquisition of a ras (Ha, Ki, or N), neu, myc, A-raf, v-raf, erbA, or erbB gene of rat origin. Several transformants' restriction enzyme sensitivity was analyzed, and their activity indicated that similar transforming sequences were present in at least two tumors and that one tumor contained two different transforming sequences. These results suggest that during hepatocarcinogenesis induced in rats by DMN-OAc, alterations in the ras gene family occur infrequently or not at all and that several different genes (which are not homologous to common oncogenes) become activated and are capable of transforming NIH 3T3 cells.     

Carcinogen specificity in the activation of transforming genes by direct-acting alkylating agents

DNAs from rat nasal and mouse skin carcinomas and fibrosarcomasinduced by the alkylating agents methylmethane sulfonate (MMS),ß-propiolactone (BPL), and dimethyl-carbamyl chloride(DMCC) were tested for their ability to transform NIH3T3 cellsby DNA transfection. Each of eight MMS-induced rat nasal carcinomasand two of five BPL-induced mouse skin tumors were positivein the transfection assay while all of four fibrosarcomas andsix carcinomas induced by DMCC were negative. Anchorage independentgrowth, tumorigenicity in nude mice, and secondary transfectionconfirmed the transformed phenotype of the positive transfectants.The transfectants from MMS-induced tumor DNAs did not containrestriction fragments homologous to rat H-, K- or N-ras oncogenesalthough exogenous (rat) tumor-derived DNA sequences were detectedin transfectant genomes by Southern analysis. In contrast aBPL-induced mouse skin tumor showed evidence of containing activatedH-ras. These results suggest specificity among causal carcinogensfor activation of transforming genes in experimental tumors.     

ras gene activation in a minor proportion of the blast population in acute myeloid leukemia

DNAs from 22 acute myeloid leukaemia (AML) patients were screened for activated transforming genes using NIH3T3 transfection followed by assay for tumor formation in Nude mice. In four samples an activated N-ras oncogene, and in two samples an activated Ha-ras oncogene were detected in transfectants. Synthetic oligonucleotide probes were used to characterise the mutations in the ras genes. Three samples were found to be mutated to N-ras codon 12 ASP, one to N-ras codon 13 ASP and two to Ha-ras codon 12 VAL. When the corresponding AML DNAs were screened using direct gel hybridisation, the mutant ras genes were detectable in only one case. In two AML samples (82 and 84) with very low percentage blasts (3% and 19%), the absence of mutant ras signal from direct gel hybridisation may be due to the lack of sensitivity of this technique in detecting activated ras in such small fractions of the total DNA. These results illustrate the sensitivity of the in vivo tumour assay in detecting activated ras. When DNA from one of the remaining three AML DNAs was amplified using the polymerase chain reaction method, the mutation present in the transfectant was detected. These findings suggest that even in AMLs with high percentage blasts (40%, 70% and 90%), cells containing mutant ras may comprise only a minor proportion of the major leukaemic clone.     

Detection of a low frequency of activated ras genes in human melanomas using a tumorigenicity assay

We have used an assay combining DNA-mediated gene transfer and tumorigenicity in Swiss athymic mice to look for activated ras genes in solid human sporadic melanomas. This assay can detect ras oncogenes mutated at codons 12, 13, or 61. We examined a panel of 13 independent surgical specimens of primary tumors and metastases. No H- or K-ras oncogenes were detected; an N-ras oncogene, mutated at codon 61, was identified in one of the 13 samples. No N-ras genes mutated at codon 13 were detected. Thus, the tumorigenicity assay detects a low frequency of ras gene activation in melanomas.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

N-ras mutation in ultraviolet radiation-induced murine skin cancers.

UV radiation is a potent DNA-damaging agent and a known inducer of skin cancer in experimental animals. To elucidate the role of oncogenes in UV carcinogenesis, we analyzed UV-induced murine skin tumors for mutations in codon 12, 13, or 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to UV-induced C3H mouse skin tumors, we also analyzed skin tumors induced in the same strain of mice by other carcinogenic agents such as 8-methoxypsoralen + UVA, angelicin + UVA, dimethylbenz-[a]anthracene + UV + croton oil, and 4-nitroquinoline-1-oxide. We found that 4 of 20 UV-induced skin tumors contained either C----A or A----G base substitutions at N-ras codon 61. In addition, 2 of 5 melanomas possessed a G----A transition in N-ras codon 13 and an A----T transversion in N-ras codon 61, respectively. Interestingly, none of the 8-methoxypsoralen + UVA- or angelicin + UVA-induced tumors we analyzed contained mutations in any of the ras genes. However, 1 of 4 4-nitroquinoline-1-oxide-induced tumors exhibited a G----T transversion at Ki-ras codon 12, a potential site for formation of a 4-nitroquinoline-1-oxide adduct with a guanine residue. We also found that 2 nonmelanoma tumors induced by dimethylbenz[a]anthracene + UV + croton oil contained an A----T transversion at Ha-ras codon 61 position 2, which is characteristic of most dimethylbenz[a]anthracene-induced tumors. These results suggest that UV-induced C3H mouse tumors display mutations preferentially in the N-ras oncogene. Since most N-ras mutations in UV-induced tumors occurred opposite dipyrimidine sequences (T-T or C-C), one can infer that these sites are the targets for UV-induced mutation and transformation.     

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a result of genotoxic effects of this chemical. The oncogenes most frequently detected in human pulmonary adenocarcinomas are K-ras genes. Activated K-ras genes have also been found in some human lymphomas. This suggest that activation of K-ras may be important in the induction of human pulmonary adenocarcinomas and lymphomas.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oncogene activation in human myeloid leukemia

We have studied by means of DNA-mediated gene transfer the activation of protooncogenes in human myeloid leukemias that represent various stages of myeloid differentiation. DNA from three cell lines, HL-60 (promyelocytic leukemia), Rc2a (myelomonocytic leukemia), and KG-1 (acute myeloblastic leukemia), was capable of transforming NIH/3T3 cells. Hybridization analysis indicated that, in all three tumor cell lines, the N-ras oncogene was activated. The cell lines U-937 ("histiocytic lymphoma") and K-562 (erythroblastic leukemia) yielded no transforming DNA. Fresh leukemia cells derived from an acute myelomonocytic leukemia patient and from a juvenile chronic myelogenous leukemia patient contained an activated N-ras and c-Ki-ras oncogene, respectively. DNA from some other myelogenous leukemia patients was not able to transform NIH/3T3 cells. Our results indicate that hematopoietic tumors of the myeloid lineage may contain oncogenes active in NIH/3T3 cell transformation and that, in particular, the N-ras oncogene may be activated in tumors representing various stages of maturation.