Spontaneous Lymphoma Research Articles

Dietary Restriction in Mice Beginning at One Year of Age: Effect on Life-Span and Spontaneous Cancer Incidence

Lifelong dietary restriction beginning at 3 to 6 weeks of age in rodents is known to decelerate the rate of aging, increase mean and maximum life-spans, and inhibit the occurrence of many spontaneous cancers. Little is known about the effects of dietary restriction started in middle age. In the experiments now reported, the food intake of 12- to 13-month-old mice of two long-lived strains was restricted by using nutrient-enriched diets in accordance with the concept of “undernutrition without malnutrition.” The mice on the restricted diet averaged 10 to 20% increases in mean and maximum survival times as compared with the control mice. Spontaneous lymphoma was inhibited by the food restriction. Reproduced with permission from Science . Copyright 1982 American Association for the Advancement of Science. Richard Weindruch, Roy L. Walford, Dietary Restriction in Mice Beginning at One Year of Age: Effect on Life-Span and Spontaneous Cancer Incidence. Science 215 , 1415-1418 (1982).

The combination of chemotherapy and systemic immunotherapy with soluble B7–immunoglobulin G leads to cure of murine leukemia and lymphoma and demonstration of tumor-specific memory responses

AbstractMajor mechanisms underlying poor immune responses to autologous tumor-associated antigens are overwhelming tumor kinetics and the absence of effective T-cell costimulation by antigen-presenting cells. To address these issues, leukemia and lymphoma mice were treated with the combination of chemotherapy and systemic immunotherapy with recombinant soluble murine B7–immunoglobulin G (IgG) molecules. In this report, 3 murine models were used, a radiation-induced SJL acute myeloid leukemia, a transplantable spontaneous SJL lymphoma, and the C57BL/6 EL-4 thymic lymphoma. Various treatment modalities were evaluated: single treatments with either B7-IgG or chemotherapy as well as combination therapies. The results demonstrate the following: (1) in all tumor models, the combination of chemotherapy and soluble B7-IgGs is more potent than either therapy alone, leading to cure of tumor-bearing animals; (2) the therapeutic responses are T-cell–dependent, because combined therapy is not efficacious in severe combined immunodeficient mice; (3) the rejection of tumor cells leads to the development of tumor-specific immunity, because cured mice are immune to the rejected tumor but not to a different syngeneic tumor; and (4) 51Cr release assays show that rejection of tumor cells leads to the development of very potent tumor-specific cytotoxic T-lymphocyte activity. On the basis of these results, it is proposed that chemotherapy-mediated tumor reduction, together with consequent augmented tumor-antigen presentation to activated T cells, are primary mechanisms leading to curative responses. The safety profile of the B7-IgG fusion proteins and their synergy with chemotherapy strongly suggest that the combination regimen is a promising strategy in cancer treatment.

Runx2: a novel oncogenic effector revealed by in vivo complementation and retroviral tagging.

The Runx2 (Cbfa1, Pebp2alphaA, Aml3) gene was previously identified as a frequent target for transcriptional activation by proviral insertion in T-cell lymphomas of CD2-MYC transgenic mice. We have recently shown that over-expression of the full-length, most highly expressed Runx2 isoform in the thymus perturbs T-cell development, leads to development of spontaneous lymphomas at low frequency and is strongly synergistic with Myc. To gain further insight into the relationship of Runx2 to other lymphomagenic pathways, we tested the effect of combining the CD2-Runx2 transgene either with a Pim1 transgene (E(mu)-Pim1) or with the p53 null genotype, as each of these displays independent synergy with Myc. In both cases we observed synergistic tumour development. However, Runx2 appeared to have a dominant effect on the tumour phenotype in each case, with most tumours conforming to the CD3(+), CD8(+), CD4(+/-) phenotype seen in CD2-Runx2 mice. Neonatal infection of CD2-Runx2 mice with Moloney murine leukaemia virus (Moloney MLV) also led to a dramatic acceleration of tumour onset. Analysis of known Moloney MLV target genes in these lymphomas showed a high frequency of rearrangement at c-Myc or N-Myc (82%), and a significant number at Pim1 or Pim2 (23%), and at Pal1/Gfi1 (18%). These results indicate that Runx2 makes a distinct contribution to T-cell lymphoma development which does not coincide with any of the oncogene complementation groups previously identified by retroviral tagging.

The sno gene, which encodes a component of the histone deacetylase complex, acts as a tumor suppressor in mice.

The Ski and Sno oncoproteins are components of a macromolecular complex containing the co-repressor N-CoR/SMRT, mSin3 and histone deacetylase. This complex has been implicated in the transcriptional repression exerted by a number of repressors including nuclear hormone receptors and Mad. Further more, Ski and Sno negatively regulate transforming growth factor-beta (TGF-beta) signaling by recruiting this complex to Smads. Here we show that loss of one copy of sno increases susceptibility to tumorigenesis in mice. Mice lacking sno died at an early stage of embryogenesis, and sno was required for blastocyst formation. Heterozygous (sno(+/-)) mice developed spontaneous lymphomas at a low frequency and showed an increased level of tumor formation relative to wild-type mice when challenged with a chemical carcinogen. sno(+/-) embryonic fibroblasts had an increased proliferative capacity and the introduction of activated Ki-ras into these cells resulted in neoplastic transformation. The B cells, T cells and embryonic fibroblasts of sno(+/-) mice had a decreased sensitivity to apoptosis or cell cycle arrest. These findings demonstrate that sno acts as a tumor suppressor at least in some types of cells.

Sensitivity to myc-induced apoptosis is retained in spontaneous and transplanted lymphomas of CD2-mycER mice.

To study the effects of the Myc oncoprotein in a regulatable in vivo system, we generated lines of transgenic mice in which a tamoxifen inducible Myc fusion protein (c-mycER) is expressed under the control of the CD2 locus control region. Activation of the Myc oncoprotein resulted in both proliferation and apoptosis in vivo. Lines with a high transgene copy number developed spontaneous lymphomas at low frequency, but the tumour incidence was significantly increased with tamoxifen treatment. Surprisingly, we found that cellular sensitivity to Myc-induced apoptosis was retained in tumours from these mice and in most lymphoma cell lines, even when null for p53. Resistance to Myc-induced apoptosis could be conferred on these cells by co-expression of Bcl-2. However, acquired resistance is clearly not an obligatory progression event as sensitivity to apoptosis was retained in transplanted tumours in athymic mice. In conclusion, lymphomas arising in CD2-mycER mice retain the capacity to undergo apoptosis in response to Myc activation and show no phenotypic evidence of the presence of an active dominant inhibitor.

Accelerated Appearance of Multiple B Cell Lymphoma Types in NFS/N Mice Congenic for Ecotropic Murine Leukemia Viruses

Spontaneous lymphomas occur at high frequency in NFS.V+ mice, strains congenic for ecotropic murine leukemia virus (MuLV) proviral genes and expressing virus at high titer. In the present study, a total of 703 NFS.V+ lymphomas were studied by histopathology, immunophenotypic analysis, immunoglobulin heavy chain or T cell receptor β chain rearrangements, and somatic ecotropic MuLV integrations; 90% of the lymphomas tested were of B cell lineage. Low-grade tumors included small lymphocytic, follicular, and splenic marginal zone lymphomas, while high-grade tumors comprised diffuse large-cell (centroblastic and immunoblastic types), splenic marginal zone, and lymphoblastic lymphomas. Comparison of mice of similar genetic background except for presence (NFS.V+) or absence (NFS.V−) of functional ecotropic MuLV genomes showed that NFS.V− clonal lymphomas developed at about one-half the rate of those occurring in NFS.V+ mice, and most were low-grade B cell lymphomas with extended latent periods. In NFS.V+ mice, clonal outgrowth, defined by Ig gene rearrangements, was associated with acquisition of somatic ecotropic proviral integrations, suggesting that, although generation of B cell clones can be virus independent, ecotropic virus may act to increase the rate of generation of clones and speed their evolution to lymphoma. The mechanism remains undefined, because only rare rearrangements were detected in several cellular loci previously associated with MuLV insertional mutagenesis.

Effect of intragastric application of N-methylnitrosourea in p53 knockout mice.

Nullizygous p53 knockout (p53(-/-)) mice are highly susceptible to spontaneous tumorigenesis, in particular malignant lymphomas at an early age. Heterozygous p53 knockout (p53(+/-)) mice develop spontaneous tumors less frequently but may show increased susceptibility to chemical carcinogens. In this study, p53(-/-), p53(+/-), and p53 wild-type (p53(+/+)) mice were treated with N-methylnitrosourea (MNU) by gastric intubation (5 microg/g body weight) three times per week for 5 wk, starting at 5-6 wk of age. The surviving mice were killed when they were 56-57 wk old. All eight p53(-/-) mice treated with MNU developed malignant lymphomas with a shorter latent period (mean age = 16.4+/-0.5 wk) than their spontaneous tumors (61%, at age 23.3+/-1.4 wk). In p53(+/-) mice treated with MNU, malignant lymphomas developed at a higher frequency (eight of 27, 30%) than did spontaneous lymphomas (5%). Development of sarcomas in p53(-/-) and p53(+/-) mice was also significantly enhanced by treatment with MNU. All eight thymic lymphomas and three sarcomas in the p53(+/-) mice showed a loss of the remaining wild-type p53 allele. These results indicate that intragastric MNU treatment significantly enhanced spontaneous development of malignant lymphomas and sarcomas in both p53(-/-) and p53(+/-) mice. In the stomachs of 12 p53(+/-) mice, that were killed at the end of the experiment, two adenomas, one carcinoma in situ, and four adenocarcinomas were observed. In the stomachs of 31 p53(+/+) mice, eight adenomas and one carcinoma in situ were detected. The overall incidence of tumorous changes in the stomachs of p53(+/-) (seven of 12, 58%) and p53(+/+) (nine of 31, 29%) mice were not significantly different (P = 0.090). However, adenocarcinomas invading the submucosa were observed in p53(+/-) mice (four of 12, 33%) but not in p53(+/+) mice (zero of 31; P = 0. 004), suggesting a slightly higher susceptibility to gastric carcinogenesis induced by MNU in p53(+/-) mice. Mol. Carcinog. 28:97-101, 2000.

A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc.

The Cbfa1/PEBP2 alpha A/AML3 gene plays an essential role in osteogenesis but is also expressed in the T-cell lineage where it has been implicated in lymphoma development as a target for retroviral insertional mutagenesis. As lymphoma cells with til-1 insertion express at least five distinct Cbfa1 isoforms, it is important to establish which, if any, have intrinsic oncogenic potential. We have generated transgenic mice in which the most abundant lymphoma isoform (G1/p57) is expressed under the control of the CD2 locus control region. Co-precipitation analysis of transgenic thymus revealed high levels of Cbfa1 protein in an abundant complex containing the binding cofactor Cbfb. CD2-Cbfa1-G1 mice displayed abnormal T-cell development, with a pronounced skew towards CD8 SP cells in the thymus and developed a low incidence of spontaneous lymphomas (6% at 12 months) with cells of similar phenotype. Strongly synergistic tumour development was seen when CD2-Cbfa1-G1 mice were crossed with lines carrying myc transgenes (CD2-myc or tamoxifen-regulatable CD2-mycER) and Cbfa1 was found to rescue expression of the CD2-myc transgene in pre-leukaemic mice. However, synergy did not appear to be due to a dominant block of myc-induced apoptosis by Cbfa1 as explanted primary tumours and cell lines from CD2-Cbfa1-G1/CD2-mycER mice showed accelerated death on induction with tamoxifen at similar rates to CD2-mycER controls. Moreover, thymocytes from preleukaemic CD2-Cbfa1-G1 mice showed reduced survival in vitro and increased sensitivity to the inhibitory effects of TGF-beta. This study demonstrates that a full-length Cbf alpha-chain gene can act as an oncogene without fusion to a heterologous protein.

Overexpression of Frat1 in transgenic mice leads to glomerulosclerosis and nephrotic syndrome, and provides direct evidence for the involvement of Frat1 in lymphoma progression.

The proto-oncogene Frat1 was originally identified as a common site of proviral insertion in transplanted tumors of Moloney murine leukemia virus (M-MuLV)-infected Emu-Pim1 transgenic mice. Contrary to most common insertion sites implicated in mouse T cell lymphomagenesis, retroviral insertional mutagenesis of Frat1 constitutes a relatively late event in M-MuLV-induced tumor development, suggesting that proviral activation of Frat1 contributes to progression of T cell lymphomas rather than their genesis. To substantiate this notion we have generated transgenic mice that overexpress Frat1 in various organs, including lymphoid tissues. Frat1 transgenic mice develop focal glomerulosclerosis and a nephrotic syndrome, but they do not exhibit an increased incidence of spontaneous lymphomas. Conversely, these mice are highly susceptible to M-MuLV-induced lymphomagenesis, and Frat1/Pim1 bitransgenic animals develop lymphomas with increased frequency compared to Pim1 transgenic littermates. These data support a role for Frat1 in tumor progression.

Transgenic expression of human MGMT blocks the hypersensitivity of PMS2-deficient mice to low dose MNU thymic lymphomagenesis.

Mice deficient in the DNA mismatch repair (MMR) gene, PMS2, develop spontaneous thymic lymphomas and sarcomas. We have previously shown that PMS2(-/-) mice were hypersensitive to a single i.p. injection of 50 mg/kg of N-methyl-N-nitrosourea (MNU) for thymic lymphoma induction. We postulated that MNU sensitivity was due to formation of O(6)-methylguanine (O(6)-mG), which, if unrepaired by O(6)-alkylguanine DNA alkyltransferase (AGT), leads to apoptosis in MMR competent cells and O(6)-mG:T mismatches in MMR deficient cells. Tumor induction is less in MMR(+/+) mice because cells with residual DNA adducts die, whereas mutagenized cells survive in MMR(-/-) mice. Overexpression of AGT (encoded by the methylguanine DNA methyltransferase-MGMT-gene) is known to block MNU induced tumorigenesis in mice with functional MMR. To further determine the sensitivity of PMS2(-/-) mice to MNU and the protective effect of hAGT overexpression, a low dose of MNU (25 mg/kg) was studied in PMS2(-/-) mice and PMS2(-/-)/hMGMT(+) mice. No thymic lymphomas were found in MNU-treated PMS2(+/+) and PMS2(+/-) mice. At 1 year, 46% of the MNU-treated PMS2(-/-) mice developed thymic lymphoma, compared with an incidence of 25% in both untreated PMS2(-/-) mice and MNU treated PMS2(-/-)/hMGMT(+) mice. In addition, a significantly shorter latency in the onset of thymic lymphomas was seen in MNU-treated PMS2(-/-) mice. K-ras mutations were detected almost equally in the thymic lymphomas induced by MNU in both PMS2(-/-) and PMS2(-/-)/hMGMT(+) mice, but not in the spontaneous lymphomas. These data suggest that PMS(-/-) mice are hypersensitive to MNU, that there are different pathways responsible for spontaneous and MNU induced thymic lymphomas in PMS2(-/-) mice, and that overexpression of hMGMT protects the mice by blocking non-K-ras pathways.

Mice defective in the DNA mismatch gene PMS2 are hypersensitive to MNU induced thymic lymphoma and are partially protected by transgenic expression of human MGMT.

DNA mismatch repair (MMR) stabilizes the cellular genome. Mice defective in the MMR gene PMS2 are susceptible to spontaneous thymic lymphoma and sarcomas. To determine the sensitivity of PMS2 knockout mice to environmental carcinogens and the protective effect of O6-methylguanine DNA methyltransferase (MGMT), heterozygous PMS2 knockout mice and human MGMT (hMGMT) transgenic mice were mated and the PMS2-/- and PMS2+/+ with or without hMGMT offspring were treated at 5 weeks of age with 50 mg/kg N-methyl-N-nitrosourea (MNU). MNU produces carcinogenic O6-methylguanine (O6-meG) adducts, resulting in thymic lymphoma in mice, which can be prevented in normal mice by overexpression of hMGMT. A significantly higher incidence of thymic lymphomas was observed in MNU-treated PMS2-/- mice, compared to wildtype PMS2+/+ mice (100 vs 52%; P < 0.001). The mean latency of lymphomas was also significantly shortened in PMS2-/- mice (81 vs 102 days, P < 0.01). Transgenic expression of hMGMT significantly but incompletely blocked MNU lymphomagenesis in PMS2-/- mice. The incidence of lymphomas in PMS2-/-/hMGMT+ mice was reduced to 80% (P < 0.01) and mean latency increased to 91 days (P < 0.05). Thymic lymphomagenesis was efficiently blocked in PMS2+/+/hMGMT+ mice with rapid repair of O6-meG. Since O6-meG:T mismatches in MMR+ cells may trigger mismatch repair resulting in abortive repair and cell death whereas in the absence of MMR, these mismatches are converted to A:T, we predicted that G to A point mutations in codon 12 of the K-ras gene would occur. In this study, we found G to A point mutations in codon 12 of the K-ras gene in many tumors. Thus, in MMR deficient tissues, methylating agents induce point mutations in cells with a higher rate of cell survival which together are potently carcinogenic in the thymus. These data suggest that PMS2 defective lymphomas may arise by the concerted action of environmental and perhaps endogenous methylation of DNA coupled to genomic instability.

Enhancement of spontaneous bursal lymphoma frequency by serotype 2 Marek's disease vaccine, SB-1, in transgenic and non-transgenic line 0 white leghorn chickens

A significant incidence of bursal lymphomas with long latencies was noted in transgenic breeders carrying a benign defective subgroup A avian leukosis provirus, ALVA6, in their germline and maintained free of exposure to avian retroviruses. Serotype 2 Marek's disease (MD) vaccine virus, strain SB-1, a component of the bivalent MD vaccine used to vaccinate the breeders, was suspected as a contributory factor in the increased bursal lymphoma incidence. Although these bursal lymphomas had several characteristics similar to retroviral-induced bursal lymphomas, we found no evidence of retroviral influence based on many virological, immunological and molecular tests that were performed on plasma and tumour cells. These tumours were therefore classified as spontaneous bursal lymphomas, similar to those reported for some specific pathogen-free (SPF) chicken lines. Long-term in vivo experiments in plastic isolators and carefully maintained pens with homozygous and hemizygous ALVA6 and ALVA6-free female chickens (line 0) that were either non-vaccinated, serotype 3 (herpesvirus of turkeys [HVT]; monovalent)-vaccinated, or HVT/SB-1 (bivalent) vaccinated, demonstrated that the incidence of spontaneous bursal lymphomas were significantly higher in those chickens that were vaccinated with the bivalent MD vaccine (P ⩽0.05). In addition, this incidence did not depend on the ALVA6 proviral insert since there was no significant difference in spontaneous bursal lymphoma incidence between bivalent vaccinated hemizygous ALVA6 and ALVA6-free line 0 female chickens. Thus, the increased incidence of spontaneous bursal lymphomas is correlated solely with the presence of SB-1 and is not dependent on the presence of ALVA6.

Low Frequency of Ras Gene Mutation in Spontaneous and Gamma-Ray-Induced Thymic Lymphomas of Scid Mice

Scid mice, which have a defect in the capacity to repair DNA double-strand breaks, were highly prone to the induction of thymic lymphomas after exposure to ionizing radiation; approximately 70% of mice developed lymphomas within 1 year after exposure to 1-3 Gy, whereas approximately 20% of unirradiated control mice developed lymphomas. To gain information on the possible role of Ras activation in development of thymic lymphomas in scid mice, we have examined both the frequency and the spectrum of Kras and Nras mutations in spontaneous and radiation-induced lymphomas. Neither activated Kras nor Nras genes were detected in spontaneous lymphomas, while Kras mutations increased in a dose-dependent manner in radiation-induced lymphomas. However, Kras mutations were infrequent (6% in lymphomas in mice exposed to 1 Gy, 12.5% in those exposed to 2 Gy, 16.7% in those exposed to 3 Gy), and no mutations were detected in Nras genes, suggesting that Ras mutation was not significantly involved in the development of thymic lymphomas in scid mice. Analysis of the spectrum of Kras mutations demonstrated unique mutations in both codons 13 (GGC to GAC) and 61 (CAA to CTA) in addition to the commonly identified substitution of GAT for GGT in codon 12 of Kras.

X-ray-induced lymphomagenesis in E mu-pim-1 transgenic mice: an investigation of the co-operating molecular events.

Transgenic mice overexpressing the pim-1 oncogene in their lymphoid compartment display a low incidence of spontaneous T-cell lymphomas, but are highly susceptible to point mutation-inducing genotoxic carcinogens. We show here that total body X-irradiation, which causes mainly chromosomal deletions, rearrangements and amplifications, significantly enhances lymphoma development in E mu-pim-1 transgenic mice. The X-ray-induced E mu-pim-1 and non-transgenic lymphomas have a comparable high cell turnover as shown by a relatively high S-phase fraction and a high apoptotic activity. Consistent with previous observations, in 75% of all lymphomas c-myc mRNA levels are 5- to 20-fold higher than in control, non-lymphomatous spleen/thymus. The expression of other oncogenes, which have previously found to be activated in combination with pim-1 in lymphomagenesis, such as gfi-1/pal-1, frat-1 and tiam-1, and also of the mdm-2 and mdm-x oncogenes, appeared not to be affected. Deletions and/or rearrangements of the p16INK4A and p15INK4B tumor suppressor genes were seldom observed (in three out of 92 X-ray-induced lymphomas). Strikingly, in addition to the high mRNA levels of the pim-1 transgene, the levels of the endogenous pim-1 transcripts were elevated significantly in 16% of the X-ray-induced E mu-pim-1 lymphomas compared with control spleen, even surpassing the level of the pim-1 transgene mRNA by 3- to 5-fold. In combination with previous results, which showed that the lymphoma incidence increased concordantly with higher levels of pim-1, this supports the notion that pim-1 can contribute to lymphomagenesis in a dose-dependent manner.

Ki-ras gene mutations and absence of p53 gene mutations in spontaneous and urethane-induced early lung lesions in CBA/J mice.

Ki-ras and p53 genes are involved in human lung carcinogenesis; however, the role of these genes in experimental lung tumors is not well known. In our study, the CBA/J mouse strain was used to investigate the presence of Ki-ras and p53 alterations in lung carcinogenesis of spontaneous tumors and tumors induced with high and low doses of urethane (ethyl carbamate). To study the presence of these alterations in the early stages of lung carcinogenesis and in very small lung tumors, restriction fragment length polymorphism and single-strand conformation polymorphism analyses were performed on polymerase chain reaction-amplified DNA from microdissected tumoral and normal lung samples. Ki-ras gene mutations in codons 12 and 61 were detected in all types of lung lesions, even in small and preneoplastic lesions, and their incidence increased with progression from lung hyperplasias (18%) to adenomas (75%) and to carcinomas (80%). Urethane exposure, in both high and low doses, increased the incidence of Ki-ras mutations in lung tumors, especially in adenomas. The presence of Ki-ras gene mutations in very small urethane-induced lung tumors and the absence of hyperplasias among the treated-group lesions may indicate that urethane accelerates tumoral progression. No p53 mutations were detected in exons 5-8 in any of the epithelium-derived lung tumors. Only one p53 mutation in exon 5 was found in a spontaneous lymphoma. Therefore, p53 mutations do not seem to cooperate with Ki-ras gene mutations or represent an alternative molecular pathway in murine carcinogenesis.

Effects of epidermal growth factor (EGF) on thymidine phosphorylase activity in homogenates of rat thyroid lobes incubated in vitro

A series of thymine phosphonomethoxyalkyl derivatives were evaluated for their ability to inhibit thymidine phosphorylase (dThdPase) purified from rat spontaneous T-cell lymphoma. A kinetic study of thymidine phosphorolysis catalyzed by dThdPase was performed with thymidine and/or inorganic phosphate as substrates. Data show that the substantial inhibitory effect of these acyclic nucleotide analogues is decreasing in the order of (R)-FPMPT > (S)-FPMPT ≥ (R)-HPMPT > (S)-PMPT > (S)-HPMPT > PMET ≥ (R)-PMPT. The inhibitory potency (Ki/dThdKm) of the most efficient inhibitors from this series against T-cell lymphoma enzyme is 0.0026 for (R)-FPMPT and 0.0048 for (S)-FPMPT. The studied compounds do not inhibit Escherichia coli and human enzyme and possess lower inhibitory potency against rat liver thymidine phosphorylase.

Clinical significance of natural killing activity in patients with advanced lymphoma.

Using a newly described analysis of natural killing activity employing an "individual effector/target cell ratio" according to the number of effector cells in blood, we recently determined that patients with spontaneous lymphoma regression had elevated natural killing activity prior to regression. To clarify the clinical significance of natural killing activity in patients with advanced lymphoma, a prospective study was performed at a single institution in 43 untreated patients. Survival was analyzed to detect prognostic variables. Among factors chosen initially by univariate analyses, multivariate analysis selected three prognostic factors: chemotherapy response (P < 0.0001), low-grade lymphoma (P = 0.0005), and natural killing activity (P = 0.0052). Within the chemotherapy response, natural killing activity was a unique correlative factor (P < 0.0001) selected by a multivariate regression analysis using forward selection method. In patients with advanced lymphoma, natural killing activity is a valuable prognostic factor and may also predict the response to chemotherapy.

Cloning and chromosome mapping of the feline genes p21WAF1 and p27Kip1

For investigation of the relation of cell cycle regulation with tumorigenesis in cats, we carried out molecular cloning of feline p21WAF1 and p27Kip1 cDNAs and chromosomal mapping of these genes on the cat genome. The feline p21WAF1 cDNA clone obtained in this study encoded 164 amino acids (aa) showing 83.5% and 76.8% sequence similarity with those of the human and mouse counterparts, respectively. The cat p27Kip1 cDNA clone isolated here encoded 198 aa, showing sequence similarities of 93.4% and 90.4% with its human and mouse counterparts, respectively. Using a panel of feline×rodent somatic cell hybrids, the feline CDKN1A ( p21WAF1) and CDKN1B ( p27Kip1) loci were assigned to feline chromosomes B2 and B4, respectively. Southern-blot analyses of 17 feline spontaneous leukemia and lymphoma cases using these cDNAs as probes did not reveal any rearrangements in either the p21WAF1 or the p27Kip1 gene. RT-PCR/SSCP (single strand conformation polymorphism) analysis of p27Kip1 cDNA did not uncover any amino acid substitutions in the 10 feline leukemia and lymphoma cases that were examined.

Apoptosis induced by γ‐irradiation, but not CD4 ligation, of peripheral T lymphocytes in vivo is p53‐dependent

Mice generated by homologous recombination which carry a large deletion of the p53 tumour suppressor gene have a high incidence of spontaneous Thy1-positive thymic lymphoma. Extra-thymic lymphomas are rare. Apoptosis following γ-irradiation in thymocytes from these animals in vitro is p53-dependent and there is a marked gene dose effect: heterozygotes show partial resistance to irradiation-induced cell death. Apoptosis in the T-cell zones of lymph nodes following in vivo γ-irradiation was p53-dependent, but the gene dosage effect was less marked than that noted for thymocytes. Apoptosis was induced in vivo by ligation of CD4 on the cell surface following intravenous injection of anti-CD4 monoclonal antibody. Apoptosis was counted in lymph node sections using a semi-automated morphometric system. This showed no evidence of p53 dependency. In contrast to a previous report, which used a different line of p53-deficient mice, splenocytes from p53-null mice did not differ significantly from wild-type cells with respect to in vitro proliferative activity and response to mitogenic stimulation by concanavalin A. This may be due to strain differences. Therefore, whilst p53 has a role in the deletion of lymphocytes which have acquired pathological DNA strand breaks which may lead to mutations, the results of this study imply that p53 is not involved in the control of apoptosis following engagement of surface receptors, nor in response to physiological DNA breaks and normal recombination events during T-cell ontogeny. © 1997 John Wiley & Sons, Ltd.

Apoptosis induced by gamma-irradiation, but not CD4 ligation, of peripheral T lymphocytes in vivo is p53-dependent.

Mice generated by homologous recombination which carry a large deletion of the p53 tumour suppressor gene have a high incidence of spontaneous Thy1-positive thymic lymphoma. Extra-thymic lymphomas are rare. Apoptosis following gamma-irradiation in thymocytes from these animals in vitro is p53-dependent and there is a marked gene dose effect: heterozygotes show partial resistance to irradiation-induced cell death. Apoptosis in the T-cell zones of lymph nodes following in vivo gamma-irradiation was p53-dependent, but the gene dosage effect was less marked than that noted for thymocytes. Apoptosis was induced in vivo by ligation of CD4 on the cell surface following intravenous injection of anti-CD4 monoclonal antibody. Apoptosis was counted in lymph node sections using a semi-automated morphometric system. This showed no evidence of p53 dependency. In contrast to a previous report, which used a different line of p53-deficient mice, splenocytes from p53-null mice did not differ significantly from wild-type cells with respect to in vitro proliferative activity and response to mitogenic stimulation by concanavalin A. This may be due to strain differences. Therefore, whilst p53 has a role in the deletion of lymphocytes which have acquired pathological DNA strand breaks which may lead to mutations, the results of this study imply that p53 is not involved in the control of apoptosis following engagement of surface receptors, nor in response to physiological DNA breaks and normal recombination events during T-cell ontogeny.