METHOD FOR DETERMINING ONCOGENIC POTENTIAL OF A CHEMICAL COMPOUND

Abstract: Methods are provided for determining the oncogenic potential of chemical compounds which utilizes a transgenic mouse predisposed to T-cell lymphomas. The transgenic mouse expresses a pim-1 oncogene and as a consequence is predisposed to the spontaneous onset of T-cell lymphomas. The oncogenic potential of a chemical compound is determined by administering a known dose of the chemical compound of interest to a pim-1 transgenic mouse. Thereafter, the transgenic mouse is monitored to detect the onset of a T-cell lymphoma. The time of onset of the T-cell lymphoma and the dosage of the chemical compound are compared to either the onset of spontaneous T-cell lymphomas in the pim-1 transgenic mouse or to the onset of a T-cell lymphoma in a pim-1 transgenic mouse which has been exposed to a known quantity of a carcinogenic agent. This provides an indication of the oncogenic potential of the chemical compound.

Description: METHOD FOR DETERMINING ONCOGENIC POTENTIAL OF A CHEMICAL COMPOUND FIELD OF THE INVENTION This invention relates to methods for determining the oncogenic potential of chemical compounds utilizing an in vivo system comprising a transgenic mouse predisposed to the development T-cell lymphomas. More particularly, the invention relates to a method for determining oncogenic potential of chemical compounds utilizing a pim-1 transgenic mouse. BACKGROUND OF THE INVENTION Infection of mice with Moloney murine leukemia virus (MuLV) induces T cell lymphomas after an average latency period of 150 days. In these lymphomas, the MuLV DNA is frequently found integrated into the mouse chromosomal DNA in the vicinity of the pim-1 oncogene. Cuypers, H.T. et al. Cell 37. 141-150 (1984) . In addition, thymomas (lymphocytic lymphomas of the thymus) of T-cell origin have been induced by N-methyl-N-nitrosourea in AKR mice which carry inherited copies of two types of leukemia virus (MuLV) . Warren, W. et al. (1987) Carcino enesis 8., 163-172. It is an object of the invention herein to provide methods to determine the oncogenic potential of chemical compounds. Such methods are highly reproducible, simple and quick. SUMMARY OF THE INVENTION In accordance with the above objects, methods are provided for determining the oncogenic potential of chemical compounds. such methods utilize a transgenic mouse predisposed to T-cell lymphomas. The transgenic mouse expresses a pim-1 oncogene and as a consequence is predisposed to the spontaneous onset of T-cell lymphomas. The oncogenic potential of a chemical compound is determined by administering a known dose of the chemical compound of interest to a pim-1 transgenic mouse. Thereafter, the transgenic mouse is monitored to detect the onset of a T-cell lymphoma. The time of onset of the T-cell lymphoma and the dosage of the chemical compound are compared to either the onset of spontaneous T-cell lymphomas in the pim-1 transgenic mouse or to the onset of a T-cell lymphoma in a pim-1 transgenic mouse which has been exposed to a known quantity of a carcinogenic agent. This provides an indication of the oncogenic potential of the chemical compound. This method may be repeated with a number of chemical compounds to determine the relative oncogenic potential of such compounds. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the construction of the transgene used to produce pim-1 transgenic mice. The upper panel shows the germline pim-1 genomic organization, in the middle panel the Eu-pim-1 construct (Van Lohuizen, M. et al. (1989) Cell 56, 673-682) and in the lower panel the H2K-pim-l construct. Figure 2 shows the multiple subcloning steps used to reinsert a BamHI fragment in the large EcoRI clone, used in the construction of the E μ -pim-1 construct of the present invention. Figure 3 shows partial fusion of a 2 kbp H2K promoter fragment to the Pstl site in front of the first exon, generating a 2.6 kbp Hindlll/Sacl fragment containing the H2K promoter and the first three and part of the fourth pim-1 exons. Figure 4 shows a partial sequence of the pim-1 gene. Figure 5 shows the intron/exon structure of the pim-1 gene as determined by comparison of cDNA and genomic DNA sequences, with exons indicated by blocks, coding sequences filled in, and horizontal arrows indicating the direction as well as the position of integrated proviruses in the numbered lymphomas. Figure 6 shows a partial sequence of the H2K promoter used in construction of the H2K-pim-l transgene. Figure 7 shows the partial nucleotide sequence of the E μ enhancer used in construction of the E -pim-1 transgene. Figure 8 depicts the N-ethyl-N-nitrosourea ("ENU")- induced lymphoma incidence in Eu-pim-1. H2K- im-l and control mice. Lymphoma-free survival of non- transgenic mice: without treatment (n=100) , after ENU treatment (n=64) , Lymphoma-free survival of Eμ-pim-1 transgenic mice: without treatment (n=200) , after ENU treatment (n=23) . Lymphoma-free survival of H2K-pim- 1 transgenic mice: without treatment (n=50) , after ENU treatment (n=24) . Figure 9 shows the Expression of c-myc. N-myc. pim-1. and endogenous viruses by Northern blot analysis. Lanes A-E positive and negative controls. Lanes A, B and C represents RNA isolated from spontaneous lymphomas in Eu-pim-1 transgenic mice. Lane E represents RNA isolated from a MuLV-induced lymphoma of a non-transgenic mouse. This lymphoma bares a proviral integration in the 3' untranslated region of the N-myc gene resulting in high expression of a slightly shorter transcript. Van Lohuizen, M. et al. Cell 56. 673-682 (1989) . Lane D represent RNA from a MuLV-induced lymphoma of a Eμ-pim-1 transgenic mouse. In this lymphoma c-mvc is highly overexpressed due to a proviral integration in c-mvc. Lanes denoted with a "T" represent tumor RNA isolated from pim-1 mice. The numbers 2, 3, 4, 22, 23, 29, 32, 33, 36, 41, 42, 53 and 62 correspond to.Eo-pim-1 transgenic mice, the numbers 25, 28, 30, 34, 35, 37, 45, 46, 47, 49, 51, 54 and 55 to H2K- i -l transgenic mouse but hardly expresses the transgene. Panel 1: Hybridization to a pim-1 specific probe; panel 2: hybridization to a 3' pim-1 probe that specifically detects the endogenous pim-1 transcript; panel 3: hybridization to a c-mγc specific probe; panel 4: hybridization to a N-mγc specific probe; panel 5: hybridization to a complete MuLV probe; panel 6: hybridization to an actin probe. DETAILED DESCRIPTION Transgenic mice expressing the pim-1 oncogene are predisposed to develop T-cell lymphomas but only to the extent that about 10% of the mice develop a lymphoma .within 240 days. When these mice are infected with MuLV, lymphomas develop in all mice in only 50-60 days. Van Lohuizen et al. Cell 56. 673-682 (1989) . In all these lymphomas MuLV DNA is integrated near either the c-myc or N-myc gene, suggesting that pim-1 and myc are synergistic in lymphomagenesis. Cuypers, H.T. et jil. Cell 37. 141- 150 (1984) ; Van Lohuizen et al. Cell 56. 673-682 (1989) . To determine the susceptibility of pim-1 transgenic mice to chemical carcinogens, N-ethyl-N- nitrosourea (ENU) has been tested. With a single low dose of ENU, nearly all pim-1 transgenic mice, but only 15% of nontransgenic mice, develop T-cell lymphomas within 200 days. All ENU-induced lymphomas in both pim-1 transgenic and nontransgenic mice express high levels of c-mvc mRNA. This supports the notion that pim-1 and c-myc are synergistic in lymphoma induction. Van Lohuizen et al. Cell 56. 673-682 (1989) . Pim-1 transgenic mice can also be used to test the oncogenic potential of other chemical compounds. As used herein, a "transgene" is a DNA sequence not found in nature introduced into the germline of a non-human animal by way of human intervention such as by way of injection of the transgene into the zygote, by retroviral infection of blastomeres, or by introduction of transgenes into embryonal stem cells by DNA transfeetion or by retrovirus-mediated transduction. Moreover, as used herein a "transgene" can comprise a DNA sequence encoding a pim-1 oncogene or DNA which is capable of hybridizing with DNA sequences encoding a pim-1 oncogene and which is capable of being expressed in a transgenic non-human animal. As used herein, a "pim-1 transgenic mouse" is a transgenic mouse containing a pim-1 oncogene which is expressed at least in the T-cells of the transgenic animal. The pim-1 oncogene in mouse has been identified as a unique oncogene which does not show homology with other known or putative oncogenes based on the failure of the sequences encoding the pim-1 gene to hybridize with other oncogenic DNA sequences. Cuypers, H.T. et al. (1984) Cell 37. 141-150. In addition, the a ino acid sequence of the pim-1 oncogene lias been determined. Selten, G. et al. (1986) Cell 46. 603-611. Pim-1 genes from other murine species may be used. They may be identified as those genes which are capable of hybridizing with DNA sequences encoding the known mouse pim-1 oncogene as well as those which share primary sequence homology " with the above mouse pim-1 oncogene. Once a pim-1 gene is identified, enhancers which facilitate T-cell expression such as the E^ enhancer sequence from the same or related animal species are positioned upstream or downstream from the pim-1 gene or within an intron. Other lymphoid specific enhancer elements can be used from TCR genes (α, β and putative and 5), major histocompatibility complex Class II, CD2, CD8, CD4, and genes encoding subunits from the CD3 complex, Thy-1 genes and Ig genes, including light chain genes lambda and kappa. Alternatively, T-cell specific promotor sequences such as the promoter from H2K may be operably linked to the pim-1 gene by replacing the normal pim-1 promotor sequence. Other promoters can be used as well, such as major histocompatibility complex Class I promoter elements, actin promoter, B2-microglobulin elements, and histon promoter elements. As used herein "operably linked", when describing the relationship between two DNA regions means that they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls transcription of the sequence; a ribosome binding site "is operably linked to a coding sequence if it is positioned so as to permit translation. In a preferred embodiment, long terminal repeat units (LTR's) from leukemia viruses specific to murine species may be positioned downstream from the pim-1 gene to further enhance the T-cell expression of the pim-1 gene. Alternatively, poly-adenylation sites such as the pim-1 3' region, other LTR sequences, SV40 small-t polyadenylation sites, and ø-globin polyadenylation sites can be used. The above pim-1 gene containing enhancer and/or promoter and/or LTR sequences to enhance T-cell expression of the pim-1 gene may be excised from the cloning vector to form a transgene which is used to form transgenic mice by conventional methods. The transgenic animals so formed are predisposed to the development of T-cell lymphomas. Two different pim-l transgenic mouse lines were constructed: E μ -pim-1 and H2K-pim-l mice. The constructs used to generate these mice are shown in Figure 1. The upper panel shows the germline pim-1 genomic organization, the middle panel shows the E^-pim-1 construct (Van Lohuizen, M. et al. (1989) Cell 56. 673-682) and the lower panel shows the H2K-pim-l construct. As used herein, a "carcinogenic agent" is defined as any agent that induces carcinoma. Such agents include viruses such as murine leukemia viruses (MuLV) and the like as well as chemical compounds such as N-methyl-N-nitrosourea, N-ethyl-N- nitrosourea, hexavalent chromium compounds, and other known carcinogenic compounds. Such known carcinogenic agents, in one aspect of the invention, may be used with the above transgenic mice to provide a time reference for the induction of T-cell lymphomas in the pim-1 transgenic mice. The time of T-cell lymphoma onset for chemical compounds can be determined and compared to this time reference as an indication of the oncogenic potential of that compound. In a preferred embodiment, a pim-1 transgenic mouse is used in conjunction with MuLV to determine the latency period after infection until the onset of T-cell lymphomas. In this particular system, the onset occurs approximately four weeks after inoculation with more than half of the infected population developing T-cell lymphomas by week 7 through 8 after inoculation. This latency period may be used as a first measure to compare the oncogenic potential of a chemical compound relative to MuLV. Thus, a specified dose of a chemical compound may induce T-cell lymphomas having a latency period which is greater than that of MuLV infection. The dose of such a compound can be increased or decreased so that the average latency period is the sεune as for MuLV thereby providing a measure of the oncogenic potential of the chemical compound, i.e., x milligrams per kg of transgenic animal, has the same oncogenic potential as MuLV. Alternatively, a known carcinogenic chemical compound such as ENU may be used instead of MuLV. The latency period and dose required for the production of T-cell lymphomas may then be used to correlate the oncogenic potential of the chemical compound being tested. Alternatively, the oncogenic potential of a chemical compound may be determined by the percentage of the transgenic mice developing T-cell lymphomas at a particular time after administration of a chemical compound. This time period is generally prior to the time of onset of spontaneous T-cell lymphomas in the transgenic mice. Such an analysis of oncogenic potential may be performed in conjunction with the induction of the T-cell lymphomas by known carcinogenic agents. Thus, as indicated in Figure 8, approximately 80% of the Eu-pim-1 transgenic mice develop T-cell lymphomas at approximately 100 to 110 days after birth (approximately 85 to 100 days after administration of ENU) . The oncogenic potential of a particular chemical compound may be compared to ENU by determining the percentage of T-cell lymphomas present in Eμ-pim-1 transgenic mice during this same time period. Thus, a similar dose of a chemical compound which results in approximately 40% of such transgenic mice developing T-cell lymphomas at 85 to 100 days after administration has a lower oncogenic potential than ENU. A third approach to determining oncogenic potential involves the measure of the rate of T-cell lymphoma development after onset of lymphomas. As shown in Figure 8, after the onset of T-cell lymphomas in approximately 20% of the Eμ-pim-1 mouse population, only about ten days are required for the next 60% of the mouse population to develop T-cell lymphomas in response to EMU. A known dose of a different chemical compound may cause a rate of lymphoma development which is greater or less than that induced by ENU. Such a chemical compound would therefore have an oncogenic potential which is respectively greater than or less than the ENU. In one aspect of the invention, a "chemical compound" does not include DNA or RNA but includes any chemical compound which does not cause the premature death of the transgenic animal. Thus, it is not practical to test the oncogenic potential of compounds such as cyanide, diphtheria toxin, etc. which cause the death of the transgenic animal prior to the onset of the T-cell lymphoma. Any other chemical compound. however, may be assayed for its oncogenic potential by the methods of the invention. The method of administration of a chemical compound is not critical. However, the method of administration may provide useful information as to the oncogenic potential of a chemical compound in a particular environment. Thus, those compounds which may be used as food additives may be tested for oncogenic potential by administration in the transgenic animal's food whereas those chemical compounds intended for use in an aerosol spray may be tested by administration via inhalation. However, any form of administration may be utilized including injections, IP or IM, preferably IP. Example 1 Construction of the E^-pim-1 transgene The DNA sequence of the genomic pim-1 gene is shown in Figure 2 of Selten, G. et al.. (1986) , Cell 46. 603-611, (see also Figure 4) . The overall structure of the pim-1 gene is shown in Figure 5. The sequence of the pim-1 transgene herein described has not been fully determined. To generate transgenic strains that overexpress the pim-1 gene in lymphoid tissues, a transgene was constructed containing a duplicated immunoglobulin heavy chain enhancer as described in Banerji, J. et al. (1983-) , Cell 51. 529-536, upstream of the pim-1 promoter, ' and a single MuLV long terminal repeat (LTR) unit vithin the 3' untranslated region. The E μ enhancers, present in duplicate within an 1800 bp long Xbal fragment, were inserted in a Clal site at about 460 nucleotides upstream of the transcription initiati n sites of the pim-1 gene (Selten, G. et al. (1986), Cell 46 503-511). The LTR in the 3' untranslated region was derived from a cloned proviral integration located 900 nucleotides downstream of the translation stop codon of pim-1. Id. The E μ enhancer was used to achieve a high level of transcription, since in earlier studies transgene constructs between pim-1 and proviral sequences were not expressed after germ line transfer. Insertion of the LTR in the 3' untranslated region of pim-1 served to boost expression of the transgene further and it was observed that MuLV-induced tumors with a proviral insertion within the 3' untranslated region exhibited higher mRNA levels than tumors carrying proviruses either upstream or downstream of the pim-1 gene (Selten et al. (1986) , supra) . Insertion of the MuLV LTR within the pim-1 3' untranslated region allowed the differential detection of the E^-pim-1 mRNA and endogenous pim-1 mRNA using an LTR-specific probe and a 3' terminal pim-1 probe, respectively. The pim-l transgene construct (ppG6) is depicted in Figure 1. Construction of the E^-pim-1 transgene and E^-pim-1 mouse are described in detail in Van Lohuizen et al. (1989), Cell 56. 673-682. Specifically, the starting material for construction of the E μ -pim-1 transgene was a cloned proviral MuLV integration from tumor 1 as disclosed in Cuypers, H.T. et al. (1984), Cell 3_7, 141-150. This was a 20 kbp EcoRI fragment containing the pim-l gene and a MuLV provirus integrated in the 3' untranslated region just upstream of the Hindlll site. The BamHI fragment (from 5650-6850 in the sequence shown in Selten, G. et al. (1986), Cell 46, 603-611, Figure 2, containing the proviral integration was subcloned. Using the Kpnl site which is present in the LTR, most of the proviral sequences were deleted except for a -12- complete LTR which consisted of sequences derived from the 5' and from the 3' LTR of the provirus. Via multiple subcloning steps (as shown in Figure 2) , the Ba HI fragment was reinserted in the large EcoRI (now 12 kbp) clone. The sequence of the E μ enhancer is only partially published in Banerji et aJL. (1983) Cell 33. 729-740; see also Figure 7 herein. This sequence .is present in a 900 bp Xbal fragment that is located in the large intron in the Ig heavy chain locus. The Xbal fragment was made blunt and introduced in doublet, by chance, in the Clal as described in Cell 56. 673-682, supra) . The transgene was excised from the vector by Hindlll digestion. Example 2 Construction of the H2K-pim-l transgene Construction started with a 12 kb EcoRI clone from which the proviral sequences, except one LTR, were deleted. The 12 kb EcoRI clone is the same 12 kb fragment as described in Example 1 in which a BamHI fragment as reinserted in the large EcoRI 12 kbp clone. The H2K promoter was present on a 2 kbp Hin III/Nru fragment from which only sequences up to about -700 bp are published (Kimura et al. (1986) , Cell 44. 261-272) . See Figure 6 for a partial sequence of the H2K promoter. From the 12 kbp EcoRI fragment the 2.6 kbp Sacl fragment containing the promoter region and the first three exons and part of the fourth exon were subcloned in puc. After partial digestion with Pstl and Hindlll, the 2 kbp H2K promoter fragment was fused to. the Pstl site just in front of the first exon generating a 2.6 kbp Hindlll/Sacl fragment containing the H2K promoter and the first three and part of the fourth pim-1 exons. See Figure 5. This fragment was introduced in the 12 kbp EcoRI fragment. digested with Hindlll and Sad, replacing the pim-1 promoter region. The H2K-pim-l transgene was excised from the vector by Hindlll digestion. Example 3 Generation of transgenic mice The DNA fragments described in the above examples which were used for injection into mouse zygotes, were released from the vectors described above with appropriate restriction endonucleases and purified by agarose gel electrophoresis and electroelution. The final DNA concentration was adjusted to 4 . Fertilized mouse eggs were recovered in cumulus from the oviducts of superovulated (CBA/BrA x C57BL/LIA)F1 females that had mated with Fl males several hours earlier. The DNA fragments were injected into the most accessible pronucleus of each fertilized egg essentially as described in Hogan, L.M. et al. , Manipulation of the Mouse Embryo: A Laboratory Manual. (1986) , Cold Spring Harbor, New York. After overnight culturing, two-cell stage embryos were implanted into the oviducts of 1-day pseudopregnant Fl foster animals and carried to term. Several weeks after birth of animals that had developed from microinjected eggs, total genomic DNA was prepared from tail biopsies as described in Hogan, supra. Transgenic founders were backcrossed with either (CBA/BrA x C57BL/LIA)F1 or with the C57BL/LIA parental strain. Example 4 Induction of Lymphomas by a Chemical Carcinogen Lvmphoma Induction. Offspring of crosses between heterozygous transgenic (Eo-pim-1 and H K- im-l) and C57BL/LiA mice were injected IP at day 15 after birth with 60 mg/kg of body weight ENU, freshly dissolved in PBS acidified with acetic acid to pH 6. Mice were examined every other day for lymphoma development and sacrificed when moribund and the lymphomas collected for further analysis. Northern Blot Analysis For Northern blot analysis 25μg of total RNA, prepared by the LiCl-urea method was separated on 1% agarose formaldehyde gels (Selton, G. et al. (1984) EMBO J. 3., 3215-3222) and transferred to nitran as recommended by supplier. Probes used for RNA analysis: pim-1 probe A (Cuypers, H.T. et al. (1984) Cell 37. 141-150); the 3' pim-1 probe, inserted in M13, extends from genomic map coordinate 6619 (Hindlll) to 6939 (Bglll) (Selton, G. et al. (1984) EMBO J. 3_, 3215-3222) . C-myc and N-myc probes (Cuypers, H.T. et al. (1984) Cell 37. 141-150; Van Lohuizen et aJL. (1989) Cell 56. 673-682) , MuLV probe: a total MuLV provirus probe was used (Berns, A.J.M. et al. (1980) J. Virol 36. 254-263), actin probe. Dodemont, H.J. et al. (1982) EMBO J. 1, 167-171. These probes were 32P labeled by nick translation (actin, pim-1 endogenous specific probe; hybridization conditions were as described (Cuypers, H.T. et al. (1984) Cell 37., 141-150) with addition of 1% SDS to all solutions, final wash was at O.lxSSC, 60 β C except for the MuLV probe that was washed finally at O.lxSSC, 42 β C. Detection of RAS Mutations For the detection of ras mutations DNA sequences encoding the codons 12, 13 and 61 of both K-ras and N-ras were amplified by the Polymerase Chain Reaction (Saiki, H. et al. (1986) Nature 324. 163-166) performed essentially as described in Bos, J.L. et al. (1987) Nature 327. 293-207; and Verlaan-de Vries, M. et al. (1986) Gene 50. 313-320. Results Within 240 days, 10% of the Eu-pim-1 mice developed spontaneous T-cell lymphomas whereas none of the H2K- pim-1 or the control mice did. Fl offspring from crosses between non-transgenic mice and mice heterozygous for either Eu-pim-1 or H2K-p_im-l gene, were treated with a single ENU (N-ethyl-N- nitrosourea) dose of 60 mg/kg body weight at day 15 after birth. The lymphoma incidence of ENU-treated and non-treated transgenic and non-transgenic mice is shown in Figure 8. Both the Eu-pim-1 and H2K-pim-l mice show a strongly increased incidence of lymphomas with a reduced latency period after ENU treatment as compared to non-transgenic mice. The incidence of lymphomas found in non-transgenic mice is in accordance with other studies in which ENU and N- methyl-N-nitrosourea (MNU) were used to induce lymphomas in mice. Frei, J. et aJL. Natl. Cancer Inst. 64. 845-856 (1980) . Since MuLV-induced lymphomagenesis in Eu-pim-1 transgenic mice is mediated via the proviral activation of either the c-myc or N-myc gene, the expression levels of the c-myc and N-myc genes in lymphomas induced by ENU were determined. N-myc expression was not elevated in any of these lymphomas (Figure 9, panel 4). In contrast, high levels of c- myc mRNA in lymphoma 2, 4, and 65 were still significantly elevated as compared to lane B, representing a lymphoma with a c-myc mRNA level similar to that normal spleen. The expression level of the majority of lymphomas was similar to that observed in lymphomas in which c-myc had been activated by proviral integration (Figure 9, panel 3 compare control lanes D and E with the other lanes) . The ENU-induced lymphomas were of T-cell origin as was evident from the clonal rearrangements of the T-cell receptor β chain gene (data not shown) . FACS analyses using various T and B-cell specific cell surface markers (not shown) showed that the ENU- induced lymphomas were phenotypically indistinguishable from T-cell lymphomas occurring spontaneously in pim-1 transgenic mice or lymphomas induced by HuLV in non-transgenic mice. In a portion of these'latter lymphomas no overexpression of c-myc or N-myc was found (e.g. tumors B and C in Figure 9), indicating that a high c-myc expression is not an intrinsic property of these cells. Van Lohuizen et al. Oέll 56. 673-682 (1989) . Therefore, it is unlikely that the high expression level of c-mvc in ENU-iπduced lymphomas simply reflects the differentiation or growth state of the tumor cells. Rather, ENU is either a direct or indirect cause of the high c-mvc mRNA levels. Since carcinogenic treatment can activate endogenous retroviruses, resulting in a viremia that, in turn, might activate proto-oncogenes by proviral insertion (Warren, W. et al. Carcinogenesis 8., 163-172 (1987)), we determined whether replication of endogenous retroviruses had been induced. Northern blots of lymphoma RNAs were hybridized to a probe containing an intact MuLV genome. This probe, which also hybridizes with the 2.8-kb pim-1 transcript in the transgenic strains due to the presence of an U3LTR within the transgene, showed additional hybridizing viral RNAs in some of the lymphomas (see Figure 9, panel 5). However, the level of expression was extremely low (compare lanes D and E of MuLV-induced lymphoma RNA with lanes 4, 22, 29, 41, 45, 55, 17, 61 of ENU-induced lymphoma RNAs) . In none of the lymphomas proviral insertions were found near the c-myc gene or the N-myc gene, as was the case in all MuLV-induced lymphomas in Eμ-pim- 1 traπβge ic mice. Van Lohuizen et ~ 1. EMBO J. 8., 133-136 (1989) . We conclude that the activation of endogenous retroviruses does not play a role in the ENU-induced lymphomagenesis in these pim-1 transgenic mice. As expected, high expression of the pim-1 transgenes were found in lymphomas of the E -pim-1 and H 2 K-pim-l transgenic mice. There is one exception, the lymphoma of the H2K-pim-l transgenic mouse 64 hardly expresses the transgene (Figure 9, panel 1) . Remarkably, we observed a highly variable level of endogenous pim-l expression in lymphomas of both pim-1 transgenic and non-transgenic mice (Figure 9, panel 2). It is unlikely that selection for high endogenous pim-1 expression occurs in the presence of a highly expressed pim-1 transgene. Probably, the enhanced expression of the pim-1 germline allele is a secondary effect of the (in)activation by ENU of other genes. Various studies have shown the involvement of the K-ras or N-ras. but not of H-ras in MNU-induced lymphomagenesis in mice. (Warren, W. et al. Carcinogenesis &, 163-172 (1987) ; Diamond, L.E. et al. Mol. Cell. Biol. 8 , 2233-2236 (1988) . In these studies, up to 50% of the lymphomas were found to carry mutations in codon 12 in K-ras and in codon 12, 13 and 61 in N-ras. Screening mutations in codons 12, 13, or 61 of either K- or N-ras by oligonucleotide mismatch hybridization reevaluated that in six out of twelve lymphomas of non-transgenic mice a mutation in K-ras was detected, four in codon 12 and two in codon 61. In contrast, we found only three mutations, two in K-ras codon 12 and one in N- ras codon 61, in 22 lymphomas from Eu-pim-1 mice and only one mutation, in N-ras codon 61, in 18 lymphomas of H2K-_i -l mice (see Table 1) . The lower incidence of mutations in K- or N-ras in lymphomas of pim-1 transgenic mice might be explained by a reduced selective advantage conferred by a mutation in ras in a cell already overexpressing the pim-1 transgene. Alternatively, one might argue that in both transgenic and non-transgenic mice the percentage of ras mutations with respect to number of animals treated with ENU is essentially the same (see Table 1) . Further studies using varying doses of NEU will be required to gain more insight into the interaction between c-mvc. pim-1. and ras in lymphogenesis. TABLE 1 Frequency of ras mutations in ENU-induced lymphomas in non-transgenic, H K-pim-l. and Eu-pim-1 transgenic mice. a: within a period of 240 days after ENU treatment. Six out of ten lymphomas with a ras mutation lack the normal ras allele. Complete or partial loss of the normal allele has been reported for tumors bearing a mutated neu. Bargmann C. et al. Cell 45. 649-657 (1986) ; H-ras, Quintanilla, M. et al. Nature 322. 78-80 (1986) ; and N-ras. Diamond, L.E. et εtl. Mol. Cell. Biol. 8, 2233-2236 (1986) . The observed he i- or homozygosity of the mutated allele suggests that a selective advantage is associated with the loss of the normal ras allele during lymphomagenesis. In conclusion, the above results show that pim-1 transgenic mice represent a highly sensitive in vivo system for ENU-induced lymphomagenesis. The overexpre^sion of c-myc in all ENU-induced lymphomas suggests that c-myc plays a pivotal role in the generation of these tumors. The low incidence of spontaneous tumors in pim-1 transgenic mice coupled to a nearly 100% lymphoma incidence after treatment with a single, relatively low dose of carcinogen indicates that piml transgenic mice are suitable to study the tumorigenic capacity of a diversity of chemical compounds. The foregoing is presented by way of example only and should not be construed as a limitation to the scope of permissible claims. Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention. All references are expressly incorporated herein by reference.

Claims: WHAT IS CLAIMED IS: 1. A method for determining the oncogenic potential of a chemical compound comprising: administering a known dose of said chemical compound to a pim-1 transgenic mouse. 2. The method of Claim 1 further comprising: detecting the onset of a T-cell lymphoma in said transgenic mouse, and comparing the dosage of said chemical compound and the time of onset of said T-cell lymphoma, as compared to the time of onset of a spontaneous T-cell lymphoma in a pim-1 transgenic mouse which has not been exposed to said chemical compound, as an indication of the oncogenic potential of said chemical compound. 3. The method of Claim 1 further comprising: detecting the onset, if any, of a T-cell lymphoma in said transgenic mouse, and comparing the dosage of said chemical compound and the time of onset of said T-cell lymphoma, as compared to the time of onset of a T-cell lymphoma in a pim-1 transgenic mouse which has been exposed to a known quantity of a carcinogenic agent, as an indication of the oncogenic potential of said chemical compound. 4. The method of Claim 3 wherein said carcinogenic agent is murine leukemia virus or ENU.

Inventors: BERNS ANTON J M, BERNS, ANTON, J., M.
Applicants: GENPHARM INT, GENPHARM INTERNATIONAL, INC.
Application year: 1990
Main classification: C12N15
IPC codes: A01K67, A61K49, C07K14, C12N15, C12Q1, G01N33