T lymphomagenesis is determined by a dominant host gene thymic lymphoma susceptible mouse-1 (TLSM-1) in mouse models.

Susceptibility to T lymphomas in mice is determined by a number of viral and host genetic factors. We analyzed the types and latent period of lymphomas spontaneously occurring in crosses between AKR/Ms, a T lymphoma-prone mouse strain, and SL/Kh, a pre-B lymphoma-prone strain. The incidence of T lymphomas in the F1 hybrids backcross to SL/Kh as well as F2 generation mice indicated that a dominant host gene thymic lymphoma susceptible mouse-1 (Tlsm-1) of AKR/Ms determined the type of lymphomas to be thymic. Linkage analysis with microsatellite markers assigned Tlsm-1 to the map position 61 cM from centromere of the chromosome 7. Close scrutiny of this region of AKXD recombinant inbred strains for spontaneous T lymphomas revealed the presence of Tlsm-1-like gene most likely between D7MIT71 (map position 62) and D7MIT13 (map position 70). On the other hand, a SL/Kh-derived recessive allele at a major histocompatibility complex (MHC)-linked locus accelerated development of both T and B lymphomas.

Mapping of the nu gene using congenic nude strains and in situ hybridization.

The chromosomal location of the nu gene, which is responsible for hairlessness and athymus, was determined using six DNA markers (interleukin 3 [Il-3], Myhs, Acrb, Evi-2, Mpo, and Hox-2) on mouse chromosome 11. We constructed the high-resolution physical mapping of the six DNA markers on chromosome 11 by in situ hybridization using fluorescence-labeled cosmid probes. The results indicate the order of centromere-(41cM)-Il-3-(3cM)-Myhs- (4cM)-Acrb-(6cM)-Evi-2-(3cM)-Mpo-(5cM)- Hox-2. We have used congenic nude strains and examined which of the six DNA markers were derived from the original nude mouse. We found the Evi-2 locus is linked to the nu gene in all the informative, independent congenic nude strains. From these data, we could estimate the location of the nu gene, not only genetically but also physically within a region that spans approximately 17 megabases (9 cM) between the Acrb and Mpo genes.

Formation of symbiotic complex by microenvironment-dependent mouse leukemias and thymic epithelial reticular cells.

Developing thymic leukemias of the mouse have been assumed to form symbiotic complexes with thymic microenvironments. This symbiosis is morphologically based on pseudoemperipolesis (PEMP). The mechanism of the association of microenvironment-dependent leukemia cells with thymic epithelial reticular cells (TER) was analyzed in vitro by scanning electron microscopy, microcinematography, and a quantitative assessment of PEMP. PEMP was a consequence of active locomotion of the leukemia cells, with TER passively accepting the leukemia cells "crawling" under their cytoplasm. The integrity of the cytoskeletal system of both cells was essentially required for PEMP, since cytochalasins and colchicine were highly inhibitory to PEMP. The mechanism of action of these compounds was probably dual: inhibition of the locomotive movements of the leukemia cells. A similar inhibition of PEMP was also observed with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

Genetically determined susceptibility of Fischer 344 rats to propylnitrosourea-induced thymic lymphomas.

Administration of propylnitrosourea p.o. by our protocol induced a high incidence of hematolymphatic neoplasms in all six rat strains studied. Remarkable strain differences in susceptibility to thymic lymphomas were observed. The incidence of thymic lymphomas was high in Fischer 344 (98%) and Wistar/Furth (71%) but low in Sprague-Dawley (29%), ACI/Ms (23%), Donryu (24%), and Long-Evans (10%) strains. Segregation of thymic lymphoma incidence among crosses between highly susceptible Fischer and poorly susceptible Long-Evans rats indicated that the increased susceptibility to thymic lymphomas of Fischer rats was determined by a dominant gene TIs-1 (thymic lymphoma susceptible) and that this gene was linked to the coat color loci, p and c, in Linkage Group I in the order of TIs-1 - c - p. The presence of another independently assorting dominant gene, TIs-2, was also suggested to accelerate the thymic lymphoma-genesis. Expression of the group-specific antigen of murine leukemia virus as well as infectious viruses was not detected in nine propylnitrosourea-induced thymic lymphomas of Fischer rats.

Quantitative trait loci affecting 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in the rat.

The incidence of tongue carcinomas (TCs) induced by oral administration of 4-nitroquinoline 1-oxide in rats is strain dependent. The inbred Dark-Agouti (DA) strain showed a much higher susceptibility to large mass-forming infiltrative TCs than did the Wistar-Furth (WF) strain. Our previous study (M. Kitano et al, Jpn. J. Cancer Res., 87: 1097-1101, 1996) on crosses between these two strains postulated a dominant susceptibility gene in DA and a dominant resistance gene in WF rats. The present study mapped these loci by analyzing the backcrosses to each parent with simple sequence repeat polymorphisms. Five quantitative parameters were analyzed: (a) the number of TCs > 5 mm in diameter; (b) the total number of TCs per rat; (c) the diameter of the largest TCs (DTCmax values); (d) the number of non-TC cancers per rat; and (e) and the number of cancers of any site per rat. All of these parameters were closely correlated (P < 0.0001). DA rats had a semidominant gene (Stc1) favoring the development of 4-nitroquinoline 1-oxide-induced cancers on chromosome 19, closely linked to D19Mit9. Peak linkage was observed 4 cM distal from D19Mit9, with a logarithm of the odds (lod) score of 5.72 for the number of large TCs and 6.08 for the DTCmax. On the other hand, WF rats had a semidominant gene (Rtc1) mapped between D1Mit1 and D1Mit3, approximately 20 cM from D1Mit1, with a peak lod score of 3.30 for both the number of large TCs and the DTCmax. The main effect of Rtc1 seemed to be to reduce the size of the TCs. The action of these genes was dose dependent and cooperative. The final incidence of TC in DA, WF, F1, and backcross rats seemed to be explained by combinations of genotype at these two loci. Possible candidate genes for Stc1 and Rtc1 are discussed.

To be or not to be a T-lymphomas, that is determined by a dominant gene Tlsm-1 in mouse models.

A single dominant gene Tlsm-1 was found to determine the type of spontaneous lymphomas to be T in the cross between AKR/Ms and SL/Kh. Microsatellite analysis mapped Tlsm-1 at the position 61 cM from centromere of Chr. 7. Study of this segment of T-lymphoma prone AKXD Rl strains also showed association of Tlsm-1 with T-lymphomas. On the other hand, lymphoma latency was significantly shorten by a recessive gene lla of SL/Kh. By a quantitative trait analysis, lla was located in class II gene in MHC.

Molecular and cytogenetic studies on nucleolar cistrons (rDNA) in mouse leukemia cells.

The gene dosage change of nucleolar cistrons (rDNA) in tumor cells has not been extensively studied. The present studies showed that increased dosage, as well as abnormal distribution of rDNA, was frequently associated with leukemia cells of SL/Ni and AKR mice. In normal SL cells, 37%, 39%, and 25% of rDNA was located in nucleolar organizer regions (NOR) of chromosomes #12, #18, and #19, respectively. Increase of rDNA/DNA was shown by hybridization on filter membranes in SL1, SL2, SL3, and M1 leukemia cells. Direct measurement of rDNA/DNA in G1 cells revealed an 11% increase in synchronized M1 cells. The increased rDNA dosage was explained by trisomy 12 in SL1 and SL2, the ectopic NOR of #9 in SL3, and the double t(X;19) marker chromosomes in M1. On the other hand, in normal AKR cells, 27%, 29%, and 45% of rDNA was assigned to NORs of chromosomes #15, #16, and #18, respectively. The relative rDNA distribution among NORs estimated by autoradiographic grain counting was suggested to be abnormal in AKR leukemia cells despite their normal karyotype; 36% rDNA was shown to be in chromosomes #15 and #16, respectively, by relative reduction in chromosome #18 in AKR1; the trisomy 15 explained the increased rDNA in AKR2; a relative increase was found in chromosome #15 in AKR3. These results were discussed with reference to the reported NOR involvement in chromosome translocation and amplification in tumor cells.

Immunohistochemical detection of ras p21 oncoprotein in undifferentiated and well-differentiated epithelial carcinomas of the human ovary.

Expression of ras p21 oncoproteins in human ovarian carcinomas was examined immunohistochemically by using a monoclonal antibody(clone RAS 10) with respect to the degree of their histological differentiation. To achieve this, the intensity of staining for the protein was compared between undifferentiated and well differentiated carcinomas, i.e. extreme subtypes of common epithelial carcinomas. The former was composed of 8 "solid" carcinomas and the latter, 11 serous, 8 mucinous, 4 endometrioid and 4 clear cell carcinomas. All the cases examined, including both undifferentiated and well-differentiated carcinomas, showed a positive reaction to this antibody. Staining intensity and the number of positive cells somewhat varied among the cases. Additionally, 2 cases of ovarian epithelial tumors of low malignant potential (I,MP) were stained with this antibody. Both the cases were positive, but the number of positive cells seemed to be rather less than that found in the carcinoma groups. Thus, no differences in ras p21 expression were observed between the cases examined in spite of the differences in the degree of differentiation of the epithelial ovarian carcinomas. However, the possibility remained that the number of positive cells could be an indicator of malignant potential, enabling us to distinguish LMPs from carcinomas.

Role of the thymus in propylnitrosourea-induced thymic lymphomagenesis in F344 rats.

The role of the thymus in propylnitrosourea (PNU)-induced thymic lymphomagenesis was studied in F344 rats with genetically determined high susceptibility. The thymus was absolutely required for thymic lymphomagenesis, since thymectomy prior to or after PNU treatment abolished lymphomagenesis, whereas grafting of a normal neonatal thymus before PNU treatment restored it. Exposure to PNU for 42 days resulted in the appearance of potentially lymphomatous cells first in the thymus, and overt T-lymphomas subsequently appeared. Such cells seemed to be thymus-dependent, since intrathymic transfer of the thymus cells from 42-day PNU-treated rats induced T-lymphomas much more efficiently than intravenous transfer. Further, grafting of the thymus from 42-day PNU-treated rats into thymectomized rats resulted in T-lymphomas of donor origin without additional PNU treatment. Cells from the spleen or bone marrow from the same donors did not give rise to T-lymphomas irrespective of the route of cell transfer and sublethal irradiation of the recipients. Morphologically atypical cell foci were detected first on the 28th day in the thymus and were most pronounced during the 35th-42nd days. Therefore, the thymus is the essential organ in which the early events of PNU-induced rat T-lymphomagenesis take place.

Development of pigmented scales on rat skin: relation to age, sex, strain, and hormonal effect.

Development of brownish scales on the skin was examined in seven inbred strains of laboratory rats, Rattus norvegicus (F344, WM, WF, LOU, BUF, ACI, and LEJ rats) up to 104 weeks old. Pigmented scales appeared mainly on the dorsum of the body but also on the perineum and the tail. The distribution of the scales changed with age and showed sex and strain differences. In males of all seven strains except F344, the scales on the dorsal region appeared at puberty then gradually developed and spread extensively in a strain-dependent pattern, until the rats were of adult age. The coloring gradually decreased with further aging. However, in F344 rats, the coloring did not spread after puberty, and in adult rats, it was partial and very weak. Among female rats, scales appeared only in LEJ rats when they were of adult and old age. Gonadectomy in WM and WF rats caused fading of the dorsal scales in males but slightly induced coloring in females. Androgen administration to the gonadectomized rats increased the pigmented scales in both sexes of both strains. In F344 rats, however, skin color was hardly changed by gonadectomy and/or the subsequent androgen administration in either sex. In F344 male rats, the testosterone concentration in serum was not significantly lower than that of WM and WF male rats. These results indicate that the development of brownish scales on rat skin is dependent on age, sex, strain, and androgen, and it is suggested that the ability producing the scales is genetically poor in the F344 strain.

Action site of the gene determining susceptibility to propylnitrosourea-induced thymic lymphomas in F344 rats.

To clarify whether the determining effect of the thymic lymphoma susceptible-1 (Tls-1) gene is on putative N-propyl-N-nitrosourea (PNU) target cells among T-lineage cells or on other host factors, we investigated the PNU-induced lymphomagenesis in transplantation chimeras between susceptible F344 and resistant LES strain rats. Administration of PNU to lethally irradiated (F344 x LES)F1 rats reconstituted with bone marrow cells from either F344 or LES parental rats invariably led to development of donor-origin thymic lymphomas. On the other hand, thymic lymphomas were induced in thymectomized F1 rats grafted with neonatal LES thymus, of which 4 out of 8 were of the donor origin. These observations indicate that the target cells of thymic lymphomagenesis of F344 and LES rats were equally susceptible to PNU provided they are in susceptible hosts and the LES thymus seems capable of supporting thymic lymphomagenesis, although this capability wanes with aging of the thymus. The effect of the Tls-1 gene, therefore, is neither on PNU susceptibility of the target cells nor on the capability of the thymus to support lymphomagenesis, but on other host factors either in or out of the thymus.

Accelerating effect of nude gene heterozygosity on spontaneous AKR thymic lymphomagenesis.

The effect of nude gene heterozygosity on spontaneous AKR thymic lymphomagenesis was studied by comparing female littermates of AKR/Ms-nu/+ and +/+. As previously reported in nude gene heterozygotes with genetic background other than AKR, AKR/Ms-nu/+ mice had a significantly smaller thymus than the +/+ littermates. Overall incidences of thymic lymphomas were comparable in the two genotypes, but the mean latent period for lymphoma development was significantly shorter in the nu/+ mice (266.3 +/- 11.6 days) than in the +/+ mice (319.3 +/- 7.9 days). Both genotypes of mice expressed a high level of XC+-ecotropic murine leukemia virus. Expression of xenotropic virus was more variable, but there was no consistent difference in onset of virus expression or in virus titer that could explain accelerated lymphomagenesis in the nude gene heterozygotes.

Strain difference of susceptibility to 4-nitroquinoline 1-oxide-induced tongue carcinoma in rats.

Strain difference of susceptibility to 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinomas of the tongue among Dark-Agouti, Long-Evans, Sprague-Dawley, ACI/Ms, Fischer 344, Donryu and Wistar/Furth rats was surveyed by evaluating the survival times, incidences and sizes of developed tumors as markers of susceptibility. Administration of 4NQO dissolved in drinking water induced squamous cell carcinomas in various sites of the upper digestive tract mucosa of all the experimental male and female rats of the seven strains. Regarding the mean survival times, Wistar/Furth rats survived much longer than any other strain of rats, and Dark-Agouti showed the shortest survival. The incidence of large, mass-type carcinomas of the tongue of Dark-Agouti rats was higher than in any other strain of rats, while that of Wistar/Furth rats was the lowest. Subsequently the mitotic activity and bromodeoxyuridine incorporation in the tongue epithelium of Dark-Agouti and Wistar/Furth rats were estimated after a short-term administration of 4NQO. There was a pronounced difference between the two strains of rats, because the proliferative responses of the tongue epithelium of Dark-Agouti rats to the 4NQO stimulation were much higher than those of Wistar/Furth rats. These results indicated that there are marked differences in the susceptibility to 4NQO-induced tongue carcinoma among the seven strains of rats, and that Dark-Agouti and Wistar/Furth rats could be useful as models of highly and poorly susceptible strains, respectively, for further genetic analysis.

Effect of BNU treatment on leukaemogenesis in lethally irradiated AKR mice restored with bone-marrow and spleen cells.

The leukaemogenic effect of N-butyl-N-nitrosourea (BNU) was studied in normal and thymectomized AKR mice which were lethally irradiated and restored with either bone-marrow (BM) or spleen cells from (AKR X AKR/T1ALD)F1 donors. In some instances T1ALD thymic cells were added to the restorative inoculum. It was possible to determine the origin of the leukemic cells by the metacentric marker chromosomes of T1ALD. The T- or B-cell characteristics were further ascertained by the cytotoxicity test for theta antigen and the EAC rosette test. All leukaemias whether thymic (TLS) or extra-thymic (ETL), developed from donor bone-marrow or spleen cells and never from the injected thymic cells. In non-thymectomized animals BNU increased the percentage of TLS and shortened their latency. Most of TLS which occurred after BNU treatment of BM-restored mice were theta-negative whereas the majority of TLS which occurred in controls and in spleen-restored animals were theta-positive. This suggests that during their maturation process BM-derived T precursors transit through a theta-negative compartment. This compartment does not reach a similar size during the maturation process of the spleen-derived precursors. Adding thymic cells to the restorative inoculum enhanced leukaemogenesis and suppressed theta-negative TLS in BM-restored mice. Thymectomized mice, restored either by BM or spleen, had a low incidence of ETL which was not significantly increased by BNU treatment except in the case of mice restored with spleen cells. The leukaemic cells of one ETL were theta-positive whereas all the other leukaemias had no detectable T or B marker. The percentage of ETL was higher in thymectomized mice treated with BNU alone than in those previously subjected to irradiation and restoration. These results strongly suggest that a theta-negative T precursor could be involved in extra-thymic leukaemogenesis but the possible involvement of a B precursor cannot be rule out unless experiments are carried out with specific markers of T- and B-cell sub-classes.

Production of thymic cells by mouse spleen and bone marrow.

Karyotype difference between the AKR/TIALD strain (T1) that bears 2 metacentric markers, and the AKR strain (no marker) was used to follow the thymic repopulation of lethally irradiated (AKR X T1) F1 hybrids restored by AKR bone marrow (BM) or spleen cells. Eleven days following radiation exposure, 40-50% of the thymic cells were BM-derived in the mice restored with BM cells whereas spleen-derived cells remained below 10% in those restored with spleen cells. The thymic repopulation by spleen-derived elements was enhanced either by injecting a larger munber of spleen cells or by adding thymic cells to the spleen inoculum; however in both cases the appearance of the spleen-derived karyotypes still required a delay of about 11 days. The thymic cells could either recruit thymic precursor cells or trigger their multiplication. On the opposite, it has not been possible to demonstrate a favorable effect of the injected thymic cells on the repopulation of the thymus by BM-derived elements.

Origin of leukemic cells in mouse leukemia induced by N-butylnitrosourea.

Administration of N-butylnitrosourea (BNU) induces leukemia in thymectomized C57BL/6J and C3Hf/Bi mice with almost the same high frequency as in non-thymectomized mice. Thymectomized and BNU-treated (C3Hf/Bi times CBA/H-T6T6)F1 mice receiving neonatal thymus tissues from C3Hf donors developed leukemias with or without marked enlargement of the grafts. The origin of leukemic cells was analysed by T6 marker chromosome and thymus allo-antigen theta in this hybrid system. Cells from leukemia with enlarged thymus grafts possessed the sigma-antigen detected by cytotoxicity tests. Cells from leukemia without thymus involvement had no sigma antigen. The leukemic cells arising at the site of thymus grafts were derived from the graft itself (C3Hf) or from the host (C3Hf times CBA/H-T6T6)F1 cells, most probably bone marrow cells which are repopulating into the graft. When the mice were treated with BNU after the lymphoid elements in the grafted thymus had been replaced by host cells, leukemia mainly composed of host-origin cells developed. Leukemia in which neoplastic cells in the thymus grafts were of donor origin and those in other hematopoietic tissues were of host origin was found not infrequently. The present results mean that the target cells in BNU leukemogenesis are distributed within and outside the thymus and that some leukemias are of multifocal tissue origin.

Early changes of dog esophageal mucosa induced by N-ethyl-N'-nitro-N-nitrosoguanidine.

Early changes in the esophageal mucosa of dogs induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) were studied. Seven one-year-old beagle dogs were given a solution of 250 micrograms ENNG/ml to drink ad libitum for 4 months. Three different kinds of lesions (10 erosive carcinomas, 4 slightly elevated microcarcinomas and 19 leukoplakias) were recognized. These three kinds of lesions were not located adjacent to one another, and were surrounded by almost normal stratified squamous epithelium. The foci of the carcinomas revealed an abrupt transition to normal epithelium and were considered to have arisen abruptly from normal esophageal epithelium. The histogenesis of squamous cell carcinomas of the esophagus in dogs may differ from that in man.

Propylnitrosourea-induced T-lymphomas in LEXF RI strains of rats: genetic analysis.

Oral administration of propylnitrosourea (PNU) in drinking water induces high incidence of lympho-haemopoietic malignancies in rats. Previously we reported that F344 strain rats were highly susceptible to T-lymphomas, and LE/Stm rats, to erythro- or myeloid leukaemias. For analysis of the genetic factors determining types of diseases, we have established LEXF recombinant inbred strains of rats comprising 23 substrains, each derived from intercross between F344 and LE/Stm rats. Rats of 23 LEXF substrains were given PNU, and the development of tumours was observed. The overall incidence of haemopoietic tumours ranged from 100% to 66.7%, and the fractions of T-lymphomas, from 100% to 4%, showing a continuous spectrum. Based on the genetic profile published as a strain distribution pattern table for the LEXF, we screened the potential quantitative trait loci involved in determination of the types of disease and length of the latency period. Statistical calculation was performed using the Map Manager QT software developed by Manly. Four loci, on chromosome 4, 7, 10 and 18, were suggested to associate with the T-lymphoma susceptibility and three loci, on chromosome 1, 5 and 16, with the length of the latency period. These putative loci were further examined in backcross (F344 x LE)F1 x LE. Among seven loci suggested by the recombinant inbred study, three loci, on chromosome 5, 7 and 10, were significantly associated with T-lymphomas and another locus on chromosome 1, just weakly. These observations indicate that PNU-induced lymphomagenesis is a multifactorial genetic process involving a number of loci linked with susceptibility and resistance.

A lung cancer case with numerous calcified metastatic nodules of the brain.

A case of pulmonary adenocarcinoma with numerous calcified metastatic nodules of the brain is reported. Autopsy revealed about 400 metastatic nodules in the central nervous system, most of which were calcified.