Reproducible chromosome changes of polycyclic hydrocarbon-induced rat leukemia: incidence and chromosome banding pattern.

Statistical analysis of 361 cases of primary leukemia induced in outbred Long-Evans and Sprague-Dawley rats by 7,12-dimethylbenz[a]anthracene (DMBA) and 7,8,12-trimethylbenz[a]anthracene (TMBA) showed that the incidence of trisomy of chromosome No. 2 was significantly lower with TMBA (17.8%) than with DMBA (29.3%). This tendency was reproducible in both sexes. Another characteristic chromosome abnormality, long No. 2, was found in 10 cases (2.8%). Quinacrine fluorescence analysis revealed that cells with No. 2 trisomy or either of two types of long No. 2 had total and partial No. 2 trisomy, respectively. Other chromosome members of cells with long No. 2, as well as the chromosomes of cells with typical No. 2 trisomy and "normal diploid" leukemia cells, revealed no band abnormality. The phenotype of No. 2 trisomy, severe anemia of the hosts reported in DMBA-induced leukemias, was also noted in leukemias with TMBA-induced No. 2 trisomy but not in leukemias with long No. 2.     

Establishment and chromosome studies of in vitro lines of chemically induced rat erythroblastic leukemia cells

Eight cultured lines with a normal diploid karyotype, no. 2 trisomy, and markers involving chromosome No. 2 were established from three 7,12-dimethylbenz[a]anthracene (DMBA)- and two N-butyl-N-nitrosourea (BNU)-induced erythroblastic leukemias in noninbred Long-Evans rats. Serial in vivo and in vitro passage of these cells frequently evoked karyotype changes in stemline cells. In both lines from DMBA- and BNU-induced leukemias, ordinary and translocation no. 2 trisomy cells appeared and gradually replaced the normal diploid stemline cells. Obvious secondary karyotypic changes were also recognized in the "cloned" leukemia cells. Nucleolar chromosomes such as chromosomes no. 3 and no. 12 were frequently involved in aneuploidy and translocation. One cell line from a BNU-induced leukemia did not change its normal diploid karyotype during 12 months of in vitro passage. The above preferential growth of cells with no. 2 trisomy and the related changes during in vivo and in vitro passage as well as in-colony formation in soft agar suggest that these chromosome changes are somehow associated with the growth behavior of the leukemia cells. No positive correlation was demonstrated between karyotype and dimethyl sulfoxide-induced erythroid differentiation of the leukemia cells.     

Loss of heterozygosity at the N-ras locus in 7,12-dimethylbenz[a]anthracene-induced rat leukemia

The 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat leukemia model enables scientists to analyze cell altered by carcinogens at various stages of leukemogenesis. We have reported that a consistent type of point mutation, A→T transversion at the second base in codon 61 of the N-ras gene, was present in this leukemia and that this mutation appeared in bone marrow cells as early as 48 h after a single dose of DMBA. In addition, two leukemia cell lines with the N-ras mutation had no wild-type N-ras allele. Therefore, we examined whether these alterations were essential to the DMBA-induced leukemias. In the study reported here, we confirmed the occurrence of this N-ras mutation in 18 (86%) of 21 primary leukemias and loss of the N-ras wild-type allele in 12 (67%) of 18 leukemias with the mutated N-ras. By using microsatellite markers on chromosome 2, loss of heterozygosity (LOH) at the N-ras locus was observed in eight leukemias, all of which were shown to have lost the wild-type N-ras allele by mutant-allele-specific amplification. These results suggest that LOH related to loss of the wild-type N-ras allele reproducibly occurs in leukemias with the N-ras mutation. Considering the timing of the N-ras mutation and LOH, it is likely that the N-ras mutation is induced early, and cells that have lost the wild-type N-ras allele seem to develop into leukemia. We believe that this system provides a suitable model for studying a series of genetic alterations from the earliest stage of carcinogenesis that cannot be approached in human malignancies. Mol. Carcinog. 18:206–212, 1997. © 1997 Wiley-Liss, Inc.     

N-ras Mutations in Acute Myelogenous Leukemia: A Review of the Current Literature and an update of the Southwest Oncology Group Experience

Point mutations of the N-ras proto-oncogene have been frequently detected in samples of acute myelogenous leukemia (AML). In general the N-ras point mutation has been found in approximately 25% of samples, with some studies detecting the mutation in as many of 60% of samples. In this report we review the current literature regarding N-ras mutations in AML with emphasis on the updated experience of the Southwest Oncology Group (SWOG). The SWOG study examined 55 adult AML patients prospectively enrolled in a treatment protocol and found N-ras point mutations in 8 of 55 patients (15%). These mutations were usually in codon 12, 13 or 61, but one patient had mutations in both codons 13 and 61, and another had an unusual point mutation in N-ras codon 60. The presence of the N-ras mutation was not associated with pre-treatment clinical variables, response to induction therapy, or survival, except for a higher percentage of FAB M4 subtypes among mutation-positive patients. In this paper we compare the SWOG experience to the aggregate of literature regarding N-ras mutations in AML. In general while the N-ras mutation is common in AML, there is no clear evidence that it is sufficient or necessary for leukemic transformation. The presence of the N-ras mutation in AML does not seem to identify a unique clinical subset of AML patients.     

Specific N-ras mutation in bone marrow within 48 H of 7,12-dimethylbenz[a]anthracene treatment in Huggins-Sugiyama rat leukemogenesis

7,12-Dimethylbenz[a]anthracene (DMBA)-induced leukemias in Long-Evans rats consistently have an A→T transversion at the second base of codon 61 in the N-ras gene. This mutation is also detected in the preleukemic stage. To determine when this specific N-ras mutation occurs in the early stages of leukemogenesis, we designed the mutant allele-specific amplification method, which was sensitive enough to detect one mutant cell among 10⁶ normal cells. In the study reported here, N-ras mutation was found in bone-marrow cells 2 d after a single DMBA injection and thereafter throughout the preleukemic stage. These results show that DMBA induces a specific N-ras mutation soon after one DMBA injection and that this mutation is probably the first event in DMBA leukemogenesis. © 1996 Wiley-Liss, Inc.     

Low incidence of H-, K- and N-ras oncogene mutations in cytological specimens of laryngeal tumours

Laryngeal cancer is a rare type of neoplasia, constituting approximately 2% of all human cancers. Mutations of the ras gene family is one of the main activating mechanisms in human cancer. Their involvement in head and neck cancer has been mainly demonstrated at the level of the overexpression whereas ras mutations in these cancers are rare in the Western world. In the present study we explored the incidence of codon 12-point mutation in the H-, K- and N-ras genes, in 41 laryngeal cytological specimens. These specimens corresponded to 19 benign and 22 malignant lesions of the larynx. Only two specimens carried a codon 12-point mutation in the K-ras gene (4.8%) while no mutation was detected in the H- and N-ras genes. K-ras mutations were detected in one benign and one malignant specimen. These results indicate low incidence of ras oncogene mutations in laryngeal cytological specimens.     

Cytogenetic studies on abelson-virus-induced mouse leukemias

The karyotype of Abelson-virus-induced murine leukemias was studied by G-banding. In contrast to the regular trisomy of chromosome 15 in most murine T-cell leukemias, Abelson leukemias were purely diploid, and remained diploid for up to seven consecutive passages in vivo. The hypothesis is advanced that integration into the recipient cell of the DNA copy of the large cellular insert, carried by the Abelson virus, may perform a function similar to the effects of gene duplication by trisomy in the more slowly developing murine leukemias.     

Effect of dietary curcumin and dibenzoylmethane on formation of 7,12- dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice

Female Sencar mice (6 weeks old) were administered 1 mg of 7,12- dimethylbenz[a]anthracene (DMBA) by oral gavage once a week for 5 weeks. At 20 weeks after the first dose of DMBA, 68% of mice developed mammary tumors (the average 1.08 tumors per mouse) and 45% had lymphomas/leukemias. Feeding 1% dibenzoylmethane (DBM) in AIN 76A diet, starting at 2 weeks before the first dose of DMBA and continuing until the end of the experiment, inhibited both the multiplicity and incidence of DMBA-induced mammary tumor by 97%. The incidence of lymphomas/leukemias was completely inhibited by 1% DBM diet. In contrast, feeding 2% curcumin diet had little or no effect on the incidence of mammary tumors, and the incidence of lymphomas/leukemias was reduced by 53%.      

Cytogenetic analysis of malignant skin tumors induced in chemically treated TG.AC transgenic mice

TG.AC mice (which carry a v-Ha-ras transgene) rapidly develp papillomas in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Approximately 30% of the papilomas are associated with subsequent development of maliganancies. Early-passage spindle-shaped tumor cells arising from explant cultures of TPA-induced tumors in TG-AC mice were tumorigenic when transplanted to syngeneic recipients. The v-Ha-ras transgene in the transplanted tumors was expressed at a high level. To identify possible genetic changes associated with the development of malignant tumors, explanted cells were cultured in vitro and assessed for karyotypic changes between the second and third passages by analyzing G-banded metaphase chromosomes. For comparison, skin malignancies were induced in nontransgenic FVB/N mice (parent strain) by 7, 12-dimethylbenz[a] anthracene (DMBA) Initiation and TPA promotion, and their G-banded metaphase chromosomes were analyzed. Trisomy (in at least 50% of about 30 metaphases) of chromosome 15 (in five of 15 tumors) and chromosome 6 (four of 15 tumors) was observed in TG.AC mice, independent of chemical treatment or tumor type. Of six tumors from DMBA/TPA-treated FVB/N mice, three had trisomy 10 or 15 (or both), and two appeared normal. The absence of trisomy 7 is notable because c-Ha-ras maps to that chromosome. The absence of trisomy 7 in the six FVB/N DMBA/TPA-induced skin malignancies contrasts with DMBA/TPA- induced karyotypic effects in SENCAR mice. Expression of the v-Ha-ras transgene may have precluded the requirement for endogenous mutant ras and allelic imbalance involving chromosome 7 in TG.AC mice, but it could not have in FVB/N mice. These results suggest the possibility that the observed trissmies are consequential, rather than causal, events in the development of TG.AC or FVB/N skin malignancies. Molecular genetic analysis will be required to understand the changes associated with tumorigenesis in this transgenic line as well as in the parent mouse line. ©1994 Wiley-Liss, Inc.     

Trisomy/tetrasomy 13 in seven cases of acute leukemia

We report the clinical presentation and the morphologic, histochemical, and immunophenotypic characteristics of seven patients with acute leukemia who had trisomy/tetrasomy 13 as the sole cytogenetic abnormality in their leukemia. Five patients had trisomy 13 at diagnosis of acute leukemia. All five of these patients had undifferentiated leukemias. The sixth patient, who had French-American-British (FAB) type M2 acute nonlymphocytic leukemia (ANLL), and the seventh patient with biphenotypic acute leukemia developed the trisomic clone as a new abnormality late in the course of their disease. A review of the literature revealed 28 previously reported hematologic malignancies with trisomy 13 or tetrasomy 13q as a solitary cytogenetic abnormality. Trisomy 13 appears to represent another rare but nonrandom cytogenetic abnormality in acute leukemia. In our series trisomy 13 is largely associated with acute leukemia with little myeloid or lymphoid differentiation.     

Influence of exogenous estrogen on oocytic depletion induced by 7,12-Dimethylbenz[a]anthracene in mice

The influence of exogenous estrogen on the action of 7,12-Dimethylbenz[a]anthracene (DMBA) on ovaries of Swiss albino mouse has been studied. DMBA elicits the depletion of oocytes; but concomitant administration of estrogen with DMBA reduces this loss of oocytic population significantly.     

Relationship between chemically induced Ha-ras mutation and transformation of BALB/c 3T3 cells: evidence for chemical-specific activation and cell type-specific recruitment of oncogene in transformation

BALB/c 3T3 cells were exposed to 7,12-dimethylbenz[a]anthracene (DMBA) and resultant transformed foci were analyzed for the presence of A182----T mutation at codon 61 of Ha-ras (a mutation found in many DMBA-induced animal tumors). None of the 30 independently cloned transformed cell lines contained such a mutation. In order to see whether DMBA is able to induce this mutation in BALB/c 3T3 cells, we developed a method sensitive enough to detect this specific mutation at the frequency of 10(-6). Employing this assay, we found time- and dose-dependent induction by DMBA of Ha-ras A182----T mutation in BALB/c 3T3 cells; for example, 2 wk after exposure to 100 micrograms/mL DMBA, 1.4 in 1 X 10(4) cells contained this specific mutation. On the other hand, other agents that also induce BALB/c 3T3 cell transformation, such as 3-methylcholanthrene (MCA), 12-O-tetradecanoylphorbol-13-acetate (TPA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or ultraviolet light, did not induce the mutation at detectable frequency (less than 10(-6)). These results suggest that DMBA efficiently induces Ha-ras mutation in BALB/c 3T3 cells but that this mutation is not recruited in the process of cell transformation. A hypothesis of carcinogen-specific mutation of Ha-ras gene and its tissue (cell type)-specific recruitment in carcinogenesis is proposed.     

Somatic Point Mutations in the Translocated bcl-2 Genes of Non-Hodgkin's Lvmphomasrl and Lvmphocvtic Leukemias: Imblications for Mechanisms of Tumor Progression

The t[14;18] chromosomal translocation is the most common cytogenetic abnormality found in hematolymphoid malignancies. The t[14;18] fuses the bcl-2 gene at 18q21 with the immunoglobulin heavy-chain locus at 14q32, resulting in deregulated expression ofbcl-2 and production of high levels of its encoded 26-kD protein in the majority of non-Hodgkin lymphomas. Recent data indicate that somatic point mutations frequently occur in translocated bcl-2 alleles, possibly because of the somatic hypermutation mechanism that is associated with the immunoglobulin gene loci and that normally contributes to antibody diversity. In some cases, these mutations can affect the open reading frame of the bcl-2 gene and thereby alter Bcl-2 proteins. Here, we review the currently available data about the incidence, biological effects, and possible clinical importance of somatic mutations within the translocated bcl-2 genes of human lymphomas and leukemias.     

Duplication of Philadelphia chromosome and trisomy of chromosome 21 in a pediatric patient with acute lymphoblastic leukemia

The evaluation of molecular and cytogenetic characteristics using novel techniques has significantly contributed into the insight of leukemia. In this study, immunoglobulin heavy chain gene rearrangements (V(H)D(H)J(H) region) were analyzed by polymerase chain reaction (PCR). Point mutations of the D835/I836 in the activation loop (AL) domain of the second tyrosine kinase domain of the fms-related tyrosine kinase 3 (FLT3) gene and NRAS (neuroblastoma cell line) point mutations were also analyzed by PCR. Furthermore, sequence analysis of the V(H)D(H)J(H) region was performed, as well as, chromosomal aberrations were identified by interphase fluorescence in situ hybridization (iFISH) in a 12.5-year-old boy with acute lymphoblastic leukemia. Positive MRD was found in bone marrow samples obtained at various time points during and after treatment completion prior to relapse. Molecular analysis of the FLT3 gene mutations revealed an acquired a G → T mutation at codon 835, which resulted to substitution of aspartate 835 for tyrosine (D835Y). Cytogenetic analysis with iFISH showed t(9;22) (q34;q11.2), with minor-BCR/ABL1 fusion gene in the majority of nuclei, while a subclone with duplication of the Philadelphia chromosome was observed. Triple signals of AML1 were detected in 80% of nuclei, which were compatible with trisomy of chromosome 21. BCR/ABL1 fusion gene, duplication of Philadelphia chromosome and persistence of MRD constitute inferior prognostic factors, while hyperdiploidy, trisomy of chromosome 21 and FLT3-AL mutations are related to better prognosis. The study of cytogenetic and molecular characteristics is essential in order to decide on the optimal treatment protocol in childhood leukemia.     

Variant and Masked Translocations in Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is characterized by a unique hemorrhagic syndrome, disseminated intravascular coagulation, and the association with the specific (15; 17)(q22-23;q 12-21) translocation, which disrupts the retinoic acid receptor alpha (RARA) and the promyelocytic leukemia (PML) genes. The t(15; 17) leads to the formation of two reciprocal fusion genes, PMURARA on chromosome 15 and RARA/PML on chromosome 17; it is responsible for the unique response of the disease to retinoic acid (ATRA) treatment. As was described for chronic myeloid leukemia and its associated t(9;22) [Philadelphia chromosome], variant translocations have been reported in APL, which are either complex translocations involving additional chromosome(s), or simple variant translocations involving only either one chromosome 15 or 17 and any of several chromosomes. Rearrangements of RARA and PML were documented in some of these variant translocations. In contrast, recent molecular analysis of APL cases with cytogenetically normal chromosomes 15 and 17 revealed the occurrence of submicroscopic translocations, leading to the formation of non reciprocal fusion genes, either PMURARA or RARA/PML only. Detailed analysis of such cases may shed light on the mechanisms of translocation, on the selection of oncogenic products, and on the respective role(s) of the products of the translocation. Demonstration of the existence, in some APL-like leukemias, of masked translocations with involvement of PML and RARA, thus allows to (i) confirm the diagnosis of APL, (ii) adapt the treatment and (iii) monitor the residual disease. Finally APL-like leukemias were recently reported, with either a t(11; 17) or t(5; 17), resulting in the fusion ofRARA to genes other than PML; these patients do not appear to respond to ATRA treatment. Altogether, these results emphasize the usefulness of a molecular definition of APL.     

Recurrent alterations of the short arm of chromosome 3 define a tumor suppressor region in rat mammary tumor cells.

Cytogenetic alterations associated with different stages in carcinogenesis can be distinguished in cultured human or rodent cells transformed by carcinogenic agents. Three tumorigenic rat mammary epithelial cell lines transformed in vitro with 7,12, -dimethylbenz[a]anthracene alone or in combination with 12-O-tetradecanoylphorbol-13-acetate were examined cytogenetically. Non-random alterations consisting of translocations involving the short arm of chromosome 3 and trisomy of chromosomes 14 and X were identified in all three lines. Deletion and inversion of chromosome 1 with the breakpoint at band 1q22 and a duplication 1q 32-43 and trisomy of chromosome 2 were observed in two cell lines. The accumulation of structural alterations and chromosome imbalances during the process of cell immortalization and acquisition of tumorigenicity are required for normal rat mammary cells to become malignant. Unbalanced translocations of chromosome 3 resulting in loss of the short arm had the breakpoint at 3p11. This site is a hotspot of breakage and recombination in various rat tumors and may represent a region of tumor suppressor gene critical to the development of rat mammary tumors, as well as other types of tumors.     

Trisomy 8 in human hematologic neoplasia and the c-myc and c-mos oncogenes

The c-mos and c-myc proto-oncogenes have been assigned to bands q22 and q24, respectively, of human chromosome No. 8. A gain of chromosome No. 8 is the most common abnormality observed in myeloproliferative diseases. By using probes specific for the c-mos and c-myc genes, we have analysed the genomic DNA from peripheral blood and bone marrow samples from 15 patients with various malignant myeloid diseases, including leukemia and myelodysplasia, and from one patient with non-Hodgkin's lymphoma, cell all of whom have trisomy for chromosome No. 8.

Except for one patient, the c-mos and c-myc genes were found in restriction fragments of germline size. In one patient with myelodysplasia, one c-myc allele was rearranged in a Hind III fragment, the other allele being normal. Thus, trisomy 8 associated with human hematologic neoplasia is generally not related to gross rearrangements of the c-mos or c-myc genes.