SHORT COMMUNICATION: The BN rat strain carries dominant hepatocarcinogen resistance loci

The phylogenetically distant F344 and BN rat strains and their(BNxF344) F1 hybrids were compared for susceptibility to hepatocarcinogenesisusing the ‘resistant hepatocyte’ model. Quantitativestereological analysis of frequency (number/liver) and size(mean volume and volume fraction) of placental form glutathioneS-transferase (GST-P)-positive lesions was carried out at 8,15 and 32 weeks after diethylnitrosamine initiation. The number/liverof GST-P-positive lesions at any time point was slightly higherin BN and (BNxF344)F1 rats than in F344 rats, but not statisticallydifferent. However, mean volume and volume fraction of GST-P-positivelesions were much higher in F344 than in both BN and (BNxF344)F1 rats at any time point, with a difference of up to >10-fold.GST-P-positive lesions exhibited a significantly higher labelingindex and much lower remodeling in male F344 than in BN and(BNxF344) F1 rats. HCCs were present at 54–57 weeks afterinitiation in 77% of male F344 and in no (BNxF344) F1 rats andat 70 weeks HCCs were observed in 100% of male F344 and in 23%of (BNxF344) F1 rats. These results suggest that the BN ratstrain is resistant to hepatocarcinogenesis and that its resistanceis genetically transmitted as a dominant character to F1 hybridsof the BN strain with the F344 susceptible strain.     

Resistance to BN myelogenous leukemia in rat radiation chimeras

Lewis → LBNF1 rat radiation chimeras showed marked resistance to transplanted BN myelogenous leukemia when compared to naive LBNF1, LBNF1 → LBNF1, or BN → LBNF1. This occurred in the absence of overt graft versus host disease or of anti-BN response in mixed lymphocyte culture. Bone marrow specific antigens may serve as the target of the resistance mechanism.     

Bone marrow transplantation in a BN rat model of acute myelogenous leukemia (AML)

A rat model of acute myelogenous leukemia is described. The leukemia originally induced by 7,12 dimethylbenzanthracine in the Brown Norway (BN) rat can be transferred by the inoculation of leukemic blasts intravenously into normal female BN rats. The survival of animals given 1–100 × 10⁶ leukemic blasts ranges from 15 to 25 days following tumor transfer, with the lowest number of cells injected giving the longest survival times. If 1 × 10⁶ blasts are injected and, 2 weeks later, groups of rats receive lethal doses of cyclophosphamide (CY), busulfan (BU) or total body irradiation (TBI) followed 24 h later by a syngeneic marrow graft, only animals which receive the CY conditioning survive more than 2 weeks (survival > 100 days) following the transplant. All other animals die with marrow failure (early death) or florid leukemia.Preliminary studies with an allogeneic (ACI marrow graft given to the BN rat) marrow graft suggests that leukemic rats may accept a histoincompatible graft without leukemic relapse following cyclophosphamide conditioning and postgraft immunosuppression. The anti-leukemic effect of cyclophosphamide, therefore, appears to be greater for this disease than is busulfan or total body irradiation.     

Conditions controlling long-term proliferation of Brown Norway rat promyelocytic leukemia in vitro: primary growth stimulation by microenvironment and establishment of an autonomous Brown Norway 'leukemic stem cell line'

Conditions for in vitro long-term maintenance and proliferation of the Brown Norway (BN) rat myelocytic leukemia cell (BNML) are described. During a primary culture of leukemic rat marrow, a few leukemic cells proliferated and were initially dependent on an adherent cell population but later acquired the capability of independent growth. A wild BN leukemic stem cell line has been maintained in vitro for several months, without noticeable phenotypic alterations. The doubling time of the cultured cells was 40 h. The cells were promyelocytes. The cytochemical markers of the original BN leukemia cells were preserved. The cultured cell line transferred leukemia exclusively to BN rats. Wistar and BDIX rats were resistant. The virulence of cultured leukemic cell was measured by shortened survival times after transplantation in animals of a fixed number of leukemic cells. The role of bone marrow microenvironment in the initiation of long-term growth is discussed.     

Development of a cell kinetic approach to curative therapy of acute myelocytic leukemia in remission using the cell cycle-specific drug 1-beta-D-arabinofuranosylcytosine in a rat model

The timing of sequentially administered antineoplastic drugs is one determinant of toxicity and therapeutic benefit. We have conducted a series of studies with 1-beta-D-arabinofuranosylcytosine (ara-C) in the rat model (Lewis X brown Norway F1 hybrid rats bearing brown Norway myelocytic leukemia) for human acute myelocytic leukemia to examine the factors determining optimum timing of sequential administration of this cell cycle DNA synthesis phase-specific drug. Late-stage disease in this model is not curable with ara-C, but the maximum survival is achieved by rats given serial 2-day courses of ara-C 6 days apart. ara-C given in 2- or 4-day-interval sequences to rats with late-stage disease is more toxic and not more effective. However, Lewis X brown Norway F1 hybrid rats bearing brown Norway myelocytic leukemia in early complete remission are curable with ara-C given in optimum timed sequence. In these experiments, groups of rats in early complete remission were given a 2-day course of ara-C in every-8-hr s.c. injections, and then a second 2-day course was given after 2-, 4-, 6-, 8-, 10-, or 12-day intervals. The best cure rate of rats surviving toxicity was achieved when sequentially administered 2-day courses of ara-C were given at 2- to 4-day intervals to rats in early complete remission. In the minimal residual disease state, as in late-stage disease, 2- and 4-day-interval sequencing was the most toxic. No significant number of cures of minimal residual disease could be obtained by even the maximum tolerated dose of ara-C given in longer than 6-day-interval sequences or by various continuous or intermittent schedules. The fact that the Lewis X brown Norway F1 hybrid rats bearing brown Norway myelocytic leukemia, while relatively refractory to ara-C, are curable with this drug when used in optimum timed sequence in early remission is encouraging for similar clinical trials in humans and suggests some principles for the design of such trials.     

F1 Hybrid resistance to bn rat myelogenous leukemia parallels resistance to transplantation of normal bn bone marrow

Short-term BN bone marrow engraftment, and survival after injection of BN myelogenous leukemia were measured in three different F1 hybrid strains which had BN as one parent. The two hybrids that were resistant to BN bone marrow were resistant to BNML, while the hybrid strain that accepted BN marrow died earliest after BNML injection. The results support the hypothesis that BN bone marrow and BNML share antigens that can lead to rejection of tissue grafts. The importance of such presumptive bone marrow alloantigens in the treatment of myelogenous leukemia by bone marrow transplantation is discussed.     

Human B-lymphocyte antigens expressed by lymphocytic and myelocytic leukemia cells. II. Detection by human anti-B-cell alloantisera.

The majority of human lymphocytic and myelocytic leukemia cells express a polymorphic antigen that is found on peripheral blood B-lymphocytes and cultured lymphoblastoid B-cell lines. These B-lymphocyte antigens were detected by 34 human alloantisera that were repeatedly absorbed with pooled platelets to remove all activity against HLA antigens and T-lymphocytes. Absorption studies indicated that a common antigen was present on both B-lymphocytes and positive leukemia cells. Leukemia cells could be subdivided into two groups based on the presence of the B-lymphocyte antigen. Fourteen of 18 acute myelocytic leukemia cells, 10 of 13 acute lymphoblastic leukemia cells, 4 of 6 chronic myelocytic leukemia cells, and 2 of 2 chronic lymphocytic leukemia cells were positive. This group of leukemia cells also reacted with rabbit anti-B-cell sera raised to papain digests of spleen cell membranes. F(ab')2 fragments of the rabbit antsera were shown to specifically block the reactions of the human antisera against B-cells and leukemia cells. These results suggested that the rabbit and human anti-B-cell sera were reacting with identical molecules. This conclusion was supported by immunoprecipitation data.     

Construction of a Protein-Protein Interaction Network for Chronic Myelocytic Leukemia and Pathway Prediction of Molecular Complexes

Background: Chronic myelocytic leukemia is a disease that threatens both adults and children. Great progress has been achieved in treatment but protein-protein interaction networks underlining chronic myelocytic leukemia are less known. Objective: To develop a protein-protein interaction network for chronic myelocytic leukemia based on gene expression and to predict biological pathways underlying molecular complexes in the network. Materials and Methods: Genes involved in chronic myelocytic leukemia were selected from OMIM database. Literature mining was performed by Agilent Literature Search plugin and a protein-protein interaction network of chronic myelocytic leukemia was established by Cytoscape. The molecular complexes in the network were detected by Clusterviz plugin and pathway enrichment of molecular complexes were performed by DAVID online. Results and Discussion: There are seventy-nine chronic myelocytic leukemia genes in the Mendelian Inheritance In Man Database. The protein-protein interaction network of chronic myelocytic leukemia contained 638 nodes, 1830 edges and perhaps 5 molecular complexes. Among them, complex 1 is involved in pathways that are related to cytokine secretion, cytokine-receptor binding, cytokine receptor signaling, while complex 3 is related to biological behavior of tumors which can provide the bioinformatic foundation for further understanding the mechanisms of chronic myelocytic leukemia.     

Dinaline: a new oral drug against leukemia? Preclinical studies in a relevant rat model for human acute myelocytic leukemia (BNML)

The efficacy and toxicity of Dinaline (GOE 1734; PD 104 208; NSC 328786; 4-amino-N-(2'-aminophenyl)benzamide) was evaluated in the Brown Norway acute myelocytic leukemia, which is generally accepted as a relevant preclinical model for human acute myelocytic leukemia. Upon repeated daily oral administration at least an 8 log leukemic cell kill was achieved with only less than a 1 log kill for normal pluripotent hemopoietic stem cells. Daily split-dose treatment even proved to be more effective and resulted in 40-50% cures. However, toxicity was also more pronounced in particular in regard to the gastrointestinal tract. So far, the mode of action of Dinaline is unknown, but its striking therapeutic index warrants further clinical investigation.     

Interleukin 1 production by monocytic leukemia cells and its possible role in coagulation abnormalities

Interleukin 1 (IL-1) production by leukemic cells was studied in 16 patients with myelocytic or monocytic leukemia. Leukemic cells from the patients with monocytic leukemia showed higher IL-1 production than those with myelocytic leukemia. Subclinical coagulation abnormalities were observed more frequently in patients with higher IL-1 producing monocytic leukemia. It is suggested that IL-1 from leukemic cells might play important roles in the establishment of these clinical features. Such observations might be of value in the understanding of the pathophysiology of leukemia-associated paraneoplastic phenomena.     

The Edpm5 locus prevents the 'angiogenic switch' in an estrogen-induced rat pituitary tumor

Edpm5 is one member of a group of quantitative trait loci that are responsible for the difference in susceptibility to estrogen-induced prolactinoma between the Fischer 344 (F344) and Brown Norway (BN) strains. Upon chronic estrogen treatment F344 rats develop large, hemorrhagic and invasive pituitary tumors, which exhibit both tumor angiogenesis and neoplasia. In contrast, BN rats do not develop a tumor despite an estrogen-induced increase in lactotroph density. To investigate the role of Edpm5 in the development of these tumors, we have generated a novel congenic rat strain F344.BN-Edpm5BN by introgressing the segment of rat chromosome bearing Edpm5 from BN into the F344 strain background. Phenotypic differences between F344 and F344.BN-Edpm5BN must be due to a gene(s) within the chromosomal interval encompassing Edpm5. Through use of these strains, we find that Edpm5 specifically regulates the switch to angiogenic phenotype, independent of neoplasia. The F344.BN-Edpm5BN rats developed tumors, which exhibited significant growth, 7-fold greater mass than the pituitary of untreated rats, and neoplasia indistinguishable from that of the F344 strain. However, the F344.BN-Edpm5BN rat tumor had a non-angiogenic phenotype. After chronic estrogen treatment, there was no increase in microvessel count over untreated controls in F344.BN-Edpm5BN tumors, whereas F344 rat tumors showed a significant increase (P < 0.0005). The ultrastructural morphology of the pituitary blood vessels also did not show significant angiogenesis associated changes in F344.BN-Edpm5BN rat pituitary tumors. In contrast the parental strain F344 had pronounced angiogenic activity. The F344.BN-Edpm5BN strain also fails to express VEGF at the high levels seen in the F344 rat pituitary after estrogen treatment. Hence at least one gene that has a large impact, directly or indirectly, on the switch to angiogenic phenotype must reside within the chromosomal interval that is the Edpm5 quantitative trait locus.     

Presence of a modifier gene(s) affecting early renal carcinogenesis in the Tsc2 mutant (Eker) rat model

The Eker (Tsc2 mutant) rat model of renal carcinoma is an example of Mendelian dominantly inherited predisposition to a specific cancer. Effects of genetic background on renal carcinogenesis in the Eker rat model (Eker/Eker > Eker/BN strain) indicate the presence in the BN rat genome of a modifier gene(s) that suppresses tumorigenesis. The identification of such a modifier gene(s) might help clarify the diversity of tuberous sclerosis in humans. i) We found that preneoplastic lesions in 8-week-old F1 rats [(Eker x LE) and (Eker x BN)] were more numerous in the LE strain than in the BN strain although the difference was not large. ii) We next administered N-ethyl-N-nitrosourea (ENU; single injection, i.p.) at the age of 4 weeks to amplify the strain difference in tumorigenesis, as we had done in an earlier study to identify the predisposing gene. iii) This experiment was also done in BN congenic Eker rats to confirm the strain difference in tumorigenesis. Preneoplastic lesions were fewer in BN congenic rats than in Eker rats by a factor of 100. We used this ENU system to perform a backcross experiment [F1(Eker x BN) x Eker] and finally succeeded in mapping a new modifier locus on rat chromosome 5 (the LOD score of the D5Rat12 was 3.13).     

A novel synthetic inhibitor of CDC25 phosphatases: BN82002

CDC25 dual-specificity phosphatases are essential regulators that dephosphorylate and activate cyclin-dependent kinase/cyclin complexes at key transitions of the cell cycle. CDC25 activity is currently considered to be an interesting target for the development of new antiproliferative agents. Here we report the identification of a new CDC25 inhibitor and the characterization of its effects at the molecular and cellular levels, and in animal models. BN82002 inhibits the phosphatase activity of recombinant human CDC25A, B, and C in vitro. It impairs the proliferation of tumoral cell lines and increases cyclin-dependent kinase 1 inhibitory tyrosine phosphorylation. In synchronized HeLa cells, BN82002 delays cell cycle progression at G1-S, in S phase and at the G2-M transition. In contrast, BN82002 arrests U2OS cell cycle mostly in the G1 phase. Selectivity of this inhibitor is demonstrated: (a) by the reversion of the mitotic-inducing effect observed in HeLa cells upon CDC25B overexpression; and (b) by the partial reversion of cell cycle arrest in U2OS expressing CDC25. We also show that BN82002 reduces growth rate of human tumor xenografts in athymic nude mice. BN82002 is a original CDC25 inhibitor that is active both in cell and animal models. This greatly reinforces the interest in CDC25 as an anticancer target.     

Argininosuccinate synthetase gene expression in leukemias: potential diagnostic marker for blastic crisis of chronic myelocytic leukemia.

Argininosuccinate synthetase (ASS) activity is hardly detected in human lymphocytes. In this study, we examined the ASS gene expression of various leukemia cells by a polymerase-chain-reaction method. We demonstrate here that (a) acute lymphocytic and acute myelocytic leukemia cells exhibit the highly elevated expression of the ASS gene and (b) chronic myelocytic leukemia (CML) in blastic crisis also exhibits the increase of ASS gene expression while CML in chronic phase, chronic lymphocytic leukemia and adult T leukemia cells show the similar level to that of normal lymphocytes. These results suggest that the ASS gene expression is of value as a diagnostic marker of acute type leukemia, particularly for blastic crisis of CML.     

Translocation of phospholipid-sensitive Ca2+-dependent protein kinase and its substrate, Mr 38,000 protein, in chronic myelocytic and acute myelocytic leukemias

Phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) and its substrates were investigated in neutrophils from normal subjects and in chronic myelocytic and acute myelocytic leukemic cells from patients with or without treatment for leukemia. PL-Ca-PK and its substrates were found in total particulate fraction of normal neutrophils, but less in cytosol. In leukemic cells from chronic myelocytic leukemia patients without treatment, PL-Ca-PK and its substrate, Mr 38,000 protein, increased in cytosol but decreased in total particulate fraction as compared with normal neutrophils. In leukemic cells obtained from chronic myelocytic leukemia patients after treatment mainly with busulfan, PL-Ca-PK and Mr 38,000 protein were increased in total particulate fraction but decreased in cytosol. Using leukemic cells from acute myelocytic leukemia patients with or without treatment, similar results were obtained. The change of localization of PL-Ca-PK and Mr 38,000 protein in leukemic cells appeared to be correlated to the increase or decrease of the number of leukemic cells. These results suggested that PL-Ca-PK together with the substrate, Mr 38,000 protein, might be translocated from total particulate fraction to cytosol with the onset of leukemia, and from cytosol to total particulate fraction accompanying treatment for leukemia.     

Genetic role of rat macrophage cytotoxicity against tumor.

Macrophages from the Lewis (Le) rat strain are significantly more cytotoxic to a Moloney sarcoma tumor both in vivo and in vitro, than are macrophages from the Brown Norway (BN) strain. Activity of macrophages from (Le x BN)F1 rats that are histocompatible with the Moloney sarcoma tumor is directed toward tumor and/or virus-associated antigens and is expressed as a dominant genetic trait. Experiments with backcross rats suggest that the genetic factors are unrelated to the major histocompatibility locus (AgB) of the rats. BN microphages, although not active against tumor and/or viral antigens, can become cytotoxic to cells displaying Le alloantigens.     

Effect of the interval between high dose 1-beta-D-arabinofuranosylcytosine injections on leukemic cell load, intestinal toxicity, and normal hematopoietic stem cells in a rat model for acute myelogenous leukemia

One injection of 1-beta-D-arabinofuranosylcytosine (ara-C) in BN rats bearing myelocytic leukemia induces recruitment and synchronization of the leukemic cells. A second ara-C injection, given when the largest fraction of cells is in S phase, causes the largest reduction in leukemic clonogenic cells. The relevance of recruitment and synchronization of leukemic cells after high dose ara-C (200 mg/kg) by rapid i.v. injection (comparable with 1 g/m2 in patients) has been tested in rats with respect to survival time and toxicity. Several groups of leukemic rats have been treated with seven injections of ara-C; the intervals between the injections per group were 4, 6, 9, 12, 15, 18, and 24 h, respectively. The longest mean survival time is observed in the group treated every 9 h which is 70.8 days compared to 22.6 days in nontreated leukemic controls. This 9-h interval of ara-C administration corresponds with the moment when DNA synthesis of the leukemic cells resumes after its inhibition by the ara-C. The most severe toxic side effects on the gastrointestinal system are observed in the group that received ara-C every 6 h; no toxic death has occurred in the animals treated with 15-h or longer intervals. The effect of the increasing interval between two ara-C injections on the normal hematopoietic stem cells has been measured with the colony forming unit spleen assay. This study showed that the reduction of normal stem cells due to ara-C is independent of the interval of administration. This differential effect of ara-C on leukemic and normal hematopoietic stem cell kinetics might in part explain the mechanisms of achieving a complete remission in acute leukemia.     

Ras-driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.     

Autologous stem cell transplantation for adult acute leukemia

Autologous stem cell transplantation using marrow or peripheral blood is routinely used to consolidate patients with acute myelocytic leukemia in complete remission. The situation is less clear for adult acute lymphocytic leukemia in which results achieved with all strategies are disappointing.In acute myelocytic leukemia, autografts should be done in patients with good and standard risk factors. Patients with high-risk acute myelocytic leukemia defined by poor cytogenetics or failure to achieve remission with the first induction course, should proceed to allogeneic stem cell transplantation with the best available human leukocyte antigen-identical donor (family or unrelated), and the nature of the conditioning regimen (myelo-ablative or non-myelo-ablative) should be decided in relation to age, and the patient's clinical condition. Results of autografting in acute myelocytic leukemia rely strongly on the quality of the graft. Higher doses of infused stem cells translate into lower relapse rates. Marrow purging with cyclophosphamide derivatives also diminishes the relapse incidence. Autologous stem cell transplantations using peripheral blood are presently preferred to marrow as the source of stem cells, but an aggressive prior in vivo purge (high-dose consolidation course(s) before cytaphereses) is then mandatory. In good-risk acute myelocytic leukemia, autografting is superior to high-dose ARA-C; in standard-risk acute myelocytic leukemia, both are supposedly equivalent. There is no prospective randomized study testing the two approaches in the good-standard-risk population. We presently test the combination of marrow and blood both purged by mafosfamide.In adult acute lymphocytic leukemia, good-risk patients get the best benefit from autografting over conventional chemotherapy. Maintenance chemotherapy after transplant is likely to bring benefit.Research in progress aims at facilitating access of the largest number of patients to autografting and at introducing posttransplant immunomodulation maneuvers such as tumor vaccination.     

Monoclonal gammopathy in patients with chronic and acute myeloid leukemia

Monoclonal IgG components were found in the serum of 5 of 40 patients with chronic myelocytic leukemia (12.5%), as well as in 2 of 15 patients with acute myelocytic leukemia (13.3%). These findings may represent an involvement of the lymphoplasmacytic system in myeloproliferative disorders. The significance of this association is discussed.