p53 (TRP53) deficiency-mediated antiapoptosis escape after 5 Gy X irradiation still induces stem cell leukemia in C3H/He mice: comparison between whole-body assay and bone marrow transplantation (BMT) assay

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53(+/-) (TRP53(+/-)) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53(+/-) mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.     

Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production

Resistance to intestinal amoebiasis is mouse strain dependent. C57BL/6 (B6) mice clear Entamoeba histolytica within hours of challenge, whereas C3H and CBA strains are susceptible to infection and disease. In this study, we show using bone marrow (BM) chimeric mice that mouse strain-dependent resistance is mediated by nonhemopoietic cells; specifically, B6 BM --> CBA recipients remained susceptible as measured by amoeba score and culture, whereas CBA BM --> B6 recipients remained resistant. Interestingly, hemopoietic IL-10 was required for maintaining the resistance of B6 mice, in that B6 IL-10-deficient mice and IL-10(-/-) BM --> wild-type recipients, but not IL-10(+/+) BM --> IL-10(-/-) recipients, exhibited higher amoeba scores than their wild-type controls. Additionally, C57BL/10 IL-10(-/-)Rag2(-/-) mice exhibited diminished amoeba scores and culture rates vs IL-10(-/-) mice, indicating that lymphocytes potentiated the susceptibility of IL-10-deficient mice. We conclude that nonhemopoietic cells mediate the natural resistance to intestinal amoebiasis of B6 mice, yet this resistance depends on hemopoietic IL-10 activity.     

p53-Knockout Mice Are Protected Against the Long-Term Effects of Methamphetamine on Dopaminergic Terminals and Cell Bodies

Abstract: p53-knockout mice provide a useful model to test the role of p53 in the neurotoxic effects of drugs in vivo. To test the involvement of p53 in methamphetamine (METH)-induced toxicity, wild-type mice, as well as heterozygous and homozygous p53-knockout male mice, were administered four injections of three different doses (2.5, 5.0, and 10.0 mg/kg) of the drug given at 2-h intervals within the space of 1 day. METH caused a marked dose-dependent loss of dopamine transporters in both the striatum and the nucleus accumbens of wild-type mice killed 2 weeks after drug administration. However, this METH-induced decrease in dopamine transporters was attenuated in both homozygous and heterozygous p53-knockout mice, with homozygous animals showing significantly greater protection. The possibility for p53 involvement in METH-induced toxicity was also supported by the observation that METH caused marked increases in p53-like immunoreactivity in the striata of wild-type mice and very little change in heterozygous p53-knockout mice, whereas no p53-like immunostaining was detected in the homozygous p53-knockout mice. Further support for p53 involvement was provided by the fact that METH treatment caused significant decreases in dopamine transporter mRNA and the number of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta and the ventral tegmental area of wild-type but not homozygous p53-knockout mice killed 2 weeks after cessation of METH administration. These results provide concordant evidence for a role of the tumor suppressor, p53, in the long-term deleterious effects of a drug acting on brain dopamine systems.     

Prevention of GVHD by modulation of rat bone marrow with UV-B irradiation: II. Kinetics of migration of UV-B-irradiated bone marrow cells in naive and lethally irradiated rats

UV-B irradiation (700 J/m2) of bone marrow (BM) cells prior to transplantation into lethally gamma-irradiated (1050 rad) allogeneic rats prevents the development of GVHD and results in a stable mixed lymphohematopoietic chimerism. To better understand the underlying mechanisms of the development of stable radiation chimeras in this model, this study was designed to examine whether the dose (700 J/m2) of UV-B irradiation used for the modulation of the BM inoculum would affect the homing pattern of radiolabeled BM cells compared to that of thoracic duct lymphocytes (TDL) in the naive and lethally irradiated recipients. The results showed that intravenously administered, 111Indium-oxine-labeled, unmodified TDL home specifically to the spleen, lymph nodes, and BM compartments with a subsequent recirculation of a large number of cells from the spleen to the lymph nodes. In contrast, radiolabeled, unmodified BM cells migrate specifically to the spleen, liver, and BM with the lymph nodes, thymus, and nonlymphoid organs containing very little amounts of radioactivity. The stable concentrations of radioactivity in the lymphoid and nonlymphoid compartments between 3 and 72 hr after injection suggest that BM cells, unlike TDL, do not recirculate. The migration pattern of BM cells in the naive recipient was not significantly different from that seen in lethally irradiated animals except for the higher concentration of radioactivity in the spleen and BM of irradiated animals compared to that seen in naive recipients. The similarity of tissue localization of BM cells in naive or in irradiated syngeneic recipients to that of allogeneic recipients suggests that the homing of BM cells is not MHC restricted. Our findings of similarity between tissue localization of UV-B-irradiated labeled BM cells and unmodified BM cells in naive and lethally irradiated recipients suggest that a dose of 700 J/m2 of UV-B irradiation is not capable of impairing BM cell migration although it is sufficient to abolish the homing of TDL to the HEV-bearing organs. Thus, our results show that BM cells are less susceptible to cell damage by UV-B irradiation than lymphocytes thereby providing the rationale for ex vivo modulation (rather than elimination) of mature T-lymphocytes in the donor BM inoculum with UV-B irradiation. This relatively simple and effective approach to modulation of T-cells in donor BM inoculum may be potentially useful in preventing GVHD without endangering successful engraftment in larger animals and in man.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Selective elimination of non-lpr lymphoid cells in mice undergoing lpr-mediated graft-vs-host disease

The transfer of lpr BM stem cells into lethally irradiated non-lpr recipients (including the congenic MRL/+ differing only at the lpr locus) causes GVHD characterized by a wasting syndrome. In this study we investigated the interaction between the autoimmune (lpr) and normal (A-Thy) B, T, and RBC cell lineages in two types of radiation chimeras: MRL/lpr plus A-Thy----(MRL/lpr X A-Thy)F1 and MRL/+ plus A-Thy----(MRL/lpr X A-Thy)F1. Analysis of B cell repopulation by competitive RIA of serum Igh-1 allotype showed that both the MRL and the A-Thy donor cells initially engrafted. However, by 2 to 4 mo post-transplantation the normal A-Thy allotype was barely detectable (reduced greater than 2 orders of magnitude), whereas the autoimmune MRL/lpr allotype persisted at normal levels. Similarly, investigation of the donor origin of peripheral blood T cells by two-color flow cytometry showed that by 8 mo post-transplantation normal A-Thy T cells had been eliminated and only MRL/lpr T cells were present in the circulation. In contrast, erythrocytes from both the MRL/lpr and A-Thy donor strains successfully engrafted the F1 recipients and persisted until the termination of the study. Control chimeras transplanted with a mixture of MRL/+ plus A-Thy BM were stably engrafted with both donor strains in both the erythroid and lymphoid populations. Additional experiments in which either B6/lpr or MRL/lpr (and B6/+ or MRL/+ control) BM cells were transferred into (MRL/lpr X B6/+)F1 and (MRL/lpr X B6/lpr)F1 recipients demonstrated that the development of GVHD was not simply due to increased alloreactivity by the lpr donor cells. In these chimeras only the recipients heterozygous (but not homozygous) for the lpr gene developed lpr-GVHD, although both types of recipients had identical genotypes except at the lpr locus.     

Defective transient endogenous spleen colony formation in S1/S1d mice.

WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.     

Effect of various factors on the differentiation of hematopoietic stem cells]

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.     

Ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions

Chemokines were shown to govern the trafficking of immune cells and may also play important roles in the survival and activation of these cells. We report here that under physiological conditions, the bone marrow (BM), spleen, blood and liver of Ccr5, but not of Ccr1-deficient mice, contain reduced numbers of NK cells. NK cells in the BM of Ccr5-deficient mice proliferate to a lesser extent compared to WT mice. Furthermore, spleen NK cells derived from Ccr5-deficient mice that were transplanted into irradiated recipients failed to proliferate in the host. Ccr5, but not Ccr1-deficient NK cells, failed to migrate in vitro in response to RANTES and MIP-1β but not MIP-1β or SDF-1 and had reduced activation, lower expression levels of NK cell markers and a slightly reduced capacity to adhere to target cells and stimulate their killing. Using the polyI:C mouse model for NK trafficking, we found that in the absence of Ccr5, but not Ccr1, NK cells failed to accumulate in the liver. In contrast, using the influenza viral infection as a model to evaluate NK cell proliferation, we found that Ccr5-deficient NK cells in the BM had a higher proliferation rate than WT NK cells. These results suggest a role for Ccr5 in NK cell proliferation and circulation under physiological conditions and a complex role for Ccr5 in determining the fate of NK cells under pathological conditions.     

Genetic resistance of CBA and a mice to transplanted lymphoid and hemopoietic cells of CBA.M523 mutants and their F1 hybrids

(CBA × M523)F₁, (A × M523)F₁ and M523 lymphocytes grafted into lethally irradiated CBA or A mice temporarily lose their capacity to respond to test antigens (SRBC, Vi-antigenS. typhi). Immunoresponsiveness of F₁ cells is affected to a lesser degree in lethally irradiated M523 mice. Depression of response is absent in the CBA F₁ combination, in the syngeneic combination and in CBA mice which have received transplanted cells from F₁ hybrids which do not share theM523 mutation. The number of hemopoietic (CBA × M523)F₁ colonies was also reduced in CBA mice. Resistance of CBA mice to lymphoid (CBA × M523)F₁ cells develops 18 days after birth. It can be reduced by additional recipient preirradiation or preinoculation with (CBA × M523)F₁ spleen cells. The abrogated resistance can be partially restored by CBA spleen cells. The activity of (CBA × M523)F₁ lymphocytes passaged through CBA spleen is restored in syngeneic F₁ secondary recipients but inhibited again in the CBA secondary recipients. These results are consistent with the suggestion that resistance of lethally irradiated CBA mice to hemopoietic and lymphoid (CBA × M523)F₁ cells is mediated by immunologically competent, radioresistant recipient cells rapidly reacting to transplantation antigens coded by the mutantH-2K ^ka allele. These cells temporarily suppress the functional activity of transplanted cells but do not eliminate them.     

Cryopreservation of hemopoietic tissue aimed at reducing immunological reactivity

A method has been developed of decreasing immunologic activity of lymphocytes from spleen, lymph nodes, and bone marrow (freshly collected or frozen) with a high percentage of intact stem cells. In experiments in vivo on lethally irradiated mice, it was demonstrated that during combined transplantation to the recipients of preserved bone marrow from two genetically different donors, a rapid decrease or absence of the effect of inactivation of hemopoietic stem cells under the influence of allogeneic lymphocytes was observed in the mixed graft. When it is necessary to transplant large quantities of bone marrow from several genetically different donors, the use of cryopreserved bone marrow is preferable to freshly drawn marrow due to the higher proliferative activity and the decreased risk in the development of immunological reactions.     

Effect of abrogating hybrid resistance on the survival of radiation chimeras]

A study was made of the effect of the hybrid resistance abrogation by means of the lymphoid cell administration on the survival of the lethally irradiated mice protected by the transplantation of the semiallogeneic bone marrow. Injection to the C57BLxCBA recipients of the C57BL lymphoid cells one day before the irradiation and the transplantation of the bone marrow of the same genotype (C57BL) increased the chimera survival in comparison with the untreated recipients; such pretreatment 7 days before the irradiation decreased the chimera survival. Parental spleen lymphocytes administration produced but an insignificant effect on the radioresistance both of the stem hemopoietic cells (by the endocolonisation test) and of the organism as a whole (by the 30-day survival test) of the F1 hybrid. On this basis a conclusion was drawn that the differences in the splenocyte efficacy, when they were injected at different periods before the irradiation, could not be attributed to the changes in radioresistance.     

Wild-type function of the p53 tumor suppressor protein is not required for apoptosis of mouse hepatoma cells

The role of the tumor suppressor protein p53 in apoptosis of mouse hepatoma cells was studied. Different lines were used which were either p53 wild-type or carried various types of heterozygous or homozygous p53 mutations. The presence of mutations was demonstrated to correlate with a lack in transactivating activity of p53. While UV-light effectively produced apoptosis in cells of all lines, irrespective of their p53 mutational status, gamma-irradiation induced the formation of micronuclei but failed to induce apoptosis. Both UV- and gamma-irradiation led to nuclear accumulation and increases in p53 protein in p53 wild-type cells. Similarly, no significant differences in apoptotic response between p53 wild-type and p53 mutated cells were seen with other apoptotic stimuli like CD95/APO-1/Fas or TNFalpha. These data suggest that wild-type p53 is not required for induction of apoptosis in mouse hepatoma cells which may explain the apparent lack of p53 mutations in mouse liver tumors.     

Long-term engraftment of p18INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologi-cal malignancies and congenital deficiencies. In recent years, non-myeloablative and reduced-intensity condi-tioning regimens have significantly expanded t…     

Phenomenon of parental resistance and its genetic regulation]

Lymphocytes of mice F1 (CBA X M523) and F1 (A X M523) transplanted to 1000 R irradiated CBA or A mice responded to the test antigens--SRBC or S. typhi Vi-antigen--by formation of 100--1000 times less antibody forming cells than in syngeneic recipients. An intermediate result is achieved when the lymphoid cells are transplanted to the irradiated M523 mice. Lymphocytes of mice F1 (A X CBA), F1 (CBA X C57Bl/6), or F1 (A X A.CA) developed a similar immune response in the irradiated syngeneic mice and in both parental lines. The ability of parental line M523 to respond to SRBC was the same as in the other lines studied when examined in situ or in adoptive transfer experiments. The stem hemopoietic cells of mice F1 (CBA X M523) develop in the spleen of CBA mice 2--2.5 times less hemopoietic colonies than in the spleen of syngeneic animals. A conclusion was drawn that mutation M523 in CBA mice inhibited the proliferation and differentiation of hemopoietic and lymphoid cells in the irradiated nonsyngeneic recipients.     

Elimination of porcine hemopoietic cells by macrophages in mice.

The difficulty in achieving donor hemopoietic engraftment across highly disparate xenogeneic species barriers poses a major obstacle to exploring xenograft tolerance induction by mixed chimerism. In this study, we observed that macrophages mediate strong rejection of porcine hemopoietic cells in mice. Depletion of macrophages with medronate-encapsulated liposomes (M-liposomes) markedly improved porcine chimerism, and early chimerism in particular, in sublethally irradiated immunodeficient and lethally irradiated immunocompetent mice. Although porcine chimerism in the peripheral blood and spleen of M-liposome-treated mice rapidly declined after macrophages had recovered and became indistinguishable from controls by wk 5 post-transplant, the levels of chimerism in the marrow of these mice remained higher than those in control recipients at 8 wks after transplant. These results suggest that macrophages that developed in the presence of porcine chimerism were not adapted to the porcine donor and that marrow-resident macrophages did not phagocytose porcine cells. Moreover, M-liposome treatment had no effect on the survival of porcine PBMC injected into the recipient peritoneal cavity, but was essential for the migration and relocation of these cells into other tissues/organs, such as spleen, bone marrow, and peripheral blood. Together, our results suggest that murine reticuloendothelial macrophages, but not those in the bone marrow and peritoneal cavity, play a significant role in the clearance of porcine hemopoietic cells in vivo. Because injection of M-liposomes i.v. mainly depletes splenic macrophages and liver Kupffer cells, the spleen and/or liver are likely the primary sites of porcine cell clearance in vivo.     

p53-Mediated gene activation in mice at high doses of chronic low-dose-rate γ radiation

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1-40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.     

Modification of the proliferative capacity of transplanted bone marrow colony forming units by changes in the host environment

Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation. Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within two days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57BL parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine two or four days prior to irradiation. Priming irradiation given six days before main irradiation accelerated, given two days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shordy after irradiation, rather than on the absolute numbers of CFUs available to the organism.     

Epidermal growth factor with insulin used for stimulation of exogenous colony formation in the spleen of mice irradiated with lethal doses of X-rays

Epidermal growth factor (12 ng per 1 g of body mass) and insulin (0.004 units per 1 g of body mass) were introduced into X-ray irradiated (1.8, 2.12, 2.7 Cr) mice. Four hours later bone marrow was extracted from femurs to be introduced into syngenic lethally irradiated recipients. On the 11th day after transplantation the number of exogenic spleen colonies was computed. The epidermal growth factor, in combination with insulin, stimulates in the organism the restoration of hemopoietic colony-forming cells after radiation injury.     

Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.Subject terms: Haematopoietic stem cells, Stem-cell research