Mathematical modeling of the processes of DNA synthesis in irradiated cells and cell populations. Modeling of the initiation and elongation of DNA chains in irradiated mammalian cells

Dose-dependence of DNA synthesis inhibition in cells of HeLa suspension culture, bone marrow and thymus of mice was investigated. A contribution of inhibition of replicon synthesis and DNA chain elongation to the total effect of DNA synthesis inhibition by gamma-radiation was estimated by mathematical simulation. The method is proposed for evaluation of Di that characterizes cell radiosensitivity. Radiosensitivity of HeLa cells after preirradiation or exposure to 5-fluorodeoxyuridine was assessed.     

Effect of fibroblasts from monolayer cultures of hematopoietic and lymphoid tissue on the immune response in vitro]

Stromal fibroblasts from the monolayer cultures of human bone marrow, guinea pig bone marrow, spleen, thymus and peripheral blood suppressed the response of the plagueforming cells against sheep erythrocytes in the suspension cultures of mouse spleen cells. Combined cultivation of 20 X 10(6) fibroblasts from all the mentioned sources led to complete suppression of the immune response. This suppression was less in mice immunized three days before the spleen cell explantation into the suspension cultures and was absent entirely in case the pre-immunization of spleen cell donors was accomplished nine days before the explantation.     

Components of the hematopoietic compartments in tumor stroma and tumor-bearing mice

Solid tumors are composed of cancerous cells and non-cancerous stroma. A better understanding of the tumor stroma could lead to new therapeutic applications. However, the exact compositions and functions of the tumor stroma are still largely unknown. Here, using a Lewis lung carcinoma implantation mouse model, we examined the hematopoietic compartments in tumor stroma and tumor-bearing mice. Different lineages of differentiated hematopoietic cells existed in tumor stroma with the percentage of myeloid cells increasing and the percentage of lymphoid and erythroid cells decreasing over time. Using bone marrow reconstitution analysis, we showed that the tumor stroma also contained functional hematopoietic stem cells. All hematopoietic cells in the tumor stroma originated from bone marrow. In the bone marrow and peripheral blood of tumor-bearing mice, myeloid populations increased and lymphoid and erythroid populations decreased and numbers of hematopoietic stem cells markedly increased with time. To investigate the function of hematopoietic cells in tumor stroma, we co-implanted various types of hematopoietic cells with cancer cells. We found that total hematopoietic cells in the tumor stroma promoted tumor development. Furthermore, the growth of the primary implanted Lewis lung carcinomas and their metastasis were significantly decreased in mice reconstituted with IGF type I receptor-deficient hematopoietic stem cells, indicating that IGF signaling in the hematopoietic tumor stroma supports tumor outgrowth. These results reveal that hematopoietic cells in the tumor stroma regulate tumor development and that tumor progression significantly alters the host hematopoietic compartment.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Augmentation of natural cell activity in tumor-bearing and normal mice by MVE-2

Summary Mice bearing advanced Lewis lung carcinoma were found to have significantly decreased natural killer (NK) cell activity in spleen and blood. The same pattern of lowered spontaneous NK cell activity was observed in nude mice with advanced human colon carcinoma LS 174 and in C3H mammary tumor virus-positive mice that spontaneously developed mammary adenocarcinomas. Maleic anhydride divinyl ether (MVE-2) usually augments NK cell activity in normal mice. We found that the lower level of spontaneous NK cell activity in tumor-bearing mice could be boosted by a single injection of MVE-2; however, this response was much weaker than that observed in age-matched normal mice. Multiple treatments with MVE-2 which are known to induce hyporesponsiveness to further augmentation of NK cell activity in spleen and blood of normal mice, also produced NK cell hyporesponsiveness in the spleen, bone marrow, and blood of tumor-bearing mice.Wladyslaw Budzynski is a guest researcher in the Preclinical Screening Laboratory from the Institute of Immunology and Experimental Therapy, Czerska Street 12, Wroclaw, Poland     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

EFFECTS OF BETA MERCAPTOETHANOL ON THE PROLIFERATION AND DIFFERENTIATION OF HUMAN OSTEOPROGENITOR CELLS

Antioxidants are known to influence metabolism and promote cell survival in a number of cell culture systems. However, their effects on the modulation of bone cell differentiationin vitroare not clearly defined. In the present studies we have investigated the effects of β-mercaptoethanol (βME) and ascorbate alone and in combination on human osteoprogenitors derived from bone marrow fibroblasts. In primary marrow cultures, βME stimulated colony formation (2-fold), alkaline phosphatase activity (3.5-fold) and, increased DNA synthesis (8-fold) after 21 days. Cell proliferation was increased significantly by βME during the first 4 days of a 10-day culture period, indicating stimulation of marrow osteoprogenitor proliferation. Ascorbate did not significantly augment the effects of βME in primary cultures or long-term cultures of passaged bone marrow fibroblasts. These findings indicate a potential beneficial role for βME addition for the optimal maintenance of colony formation, cell proliferation and differentiation of marrow osteoprogenitor cells in primary human bone marrow fibroblast cultures.     

Organ interactions in the regulation of hematopoiesis: in vitro interactions of bone, thymus, and spleen with bone marrow stem cells in normal, Sl/Sld and W/Wv mice.

Hematopoietic cell differentiation is influenced by organ-dependent microenvironmental factors as well as humoral regulators. A technique is described for examining certain aspects of the hemopoietic inductive microenvironment in vitro. Suspension and agar cultures of mouse bone marrow were used to study the effects of organ stromal factors on cellular proliferation and differentiation. Bone, spleen, and thymus fragments from irradiated mice were placed in direct contact with or separated by a Nuclepore membrane from syngeneic marrow cells growing in suspension cultures. Normal adult mouse bone and spleen influenced granulocytic differentiation as well as cell proliferation. In this system, bone marrow and organ fragments from W/Wv and SlSld mice behaved like those of their non-anemic littermates. The most prominent difference between W/Wv and Sl/Sla mice and their normal counterparts was observed in the inductionof CFU-C from splenic precursors un-er the influence of CSA. In both types of anemic mice, in vitro generation of CFU-C from spleen was abnormal in young animals but was corrected by four months of age.     

Influence of rimantadine, ribavirine and triazavirine on influenza A virus replication in human monolayer and lymphoblastoid cell lines

The influence of antivirals, such as rimantadine, ribavirine and triazavirine on influenza virus replication in human cell cultures was evaluated. All the antivirals inhibited viral nucleoprotein NP synthesis. The strongest effect was shown for ribavirine in lung carcinoma A-549 cells and endothelial ECV-304 cells. Hoechst-33258 staining revealed induction of apoptosis in all the cell lines. Rimantadine and ribavirine inhibited virus-induced apoptosis while ribavirine enhanced it. The effect was registered in monolayer cell cultures as well as in suspension cell cultures. The influence of the antiviral drugs on the virus-induced cell proliferation in the suspension cell cultures is also described.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

The induction of lymphokine synthesis and cell growth in IL 3-dependent cell lines using antigen-antibody complexes

Several IL 3-dependent murine bone marrow-derived cell lines can be stimulated to grow with antigen-antibody (Ag.Ab) complexes. The Ag.Ab complexes induced lymphokine gene expression and the synthesis of IL 2, GM-CSF, IL 3, and BSF-1 (IL 4). The lymphokines produced by these IL 3-dependent cells appeared to stimulate their own growth, as both IL 3 and BSF-1 (IL 4) stimulated the growth of IL 3-dependent cells. Ag.Ab complexes also stimulate the growth of primary cultures of bone marrow cells that have been previously activated with IL 3. Normal bone marrow, IL 2-, and GM-CSF-dependent bone marrow cell lines could bind Ag.Ab complexes, but binding did not result in the induction of lymphokine synthesis or cell growth. Hyperimmune serum from mice also stimulated lymphokine synthesis and cell growth in IL 3-dependent cells, and the stimulatory activity was removed by treatment with Staphylococcus aureus protein A, suggesting the presence of Ag.Ab complexes.     

Depletion of CD4 and CD8 T lymphocytes in mice in vivo enhances 1,25-dihydroxyvitamin D3-stimulated osteoclast-like cell formation in vitro by a mechanism that is dependent on prostaglandin synthesis

To investigate the role of T lymphocytes in osteoclastogenesis, we performed in vivo depletion of CD4 and/or CD8 T lymphocyte subsets and evaluated in vitro osteoclast-like cell (OCL) formation. T lymphocyte depletion (TLD) with mAbs was confirmed 24 h later by flow cytometry. OCL formation was stimulated with 1, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in bone marrow and with recombinant mouse (rm) receptor activator of NF-kappaB ligand (RANK-L) and rmM-CSF in bone marrow and spleen cell cultures. OCL formation was up to 2-fold greater in 1,25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice than in those from intact mice. In contrast, TLD did not alter OCL formation in bone marrow or spleen cell cultures that were stimulated with rmRANK-L and rmM-CSF. The effects of TLD seemed to be mediated by enhanced PG synthesis, because the PGE(2) concentration in the medium of 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice was 5-fold higher than that in cultures from intact mice, and indomethacin treatment abolished the stimulatory effect of TLD on OCL formation. There was a 2-fold increase in RANK-L expression and an almost complete suppression of osteoprotegerin expression in 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice compared with those from intact mice. Although there was a small (20%) increase in IL-1alpha expression in 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice, TLD in mice lacking type I IL-1R and wild-type mice produced similar effects on OCL formation. Our data demonstrate that TLD up-regulates OCL formation in vitro by increasing PG production, which, in turn, produces reciprocal changes in RANK-L and osteoprotegerin expression. These results suggest that T lymphocytes influence osteoclastogenesis by altering bone marrow stromal cell function.     

Suppression of the initiation of DNA synthesis as an index of cellular radiosensitivity

A gamma-radiation dose (Di) suppressing DNA synthesis initiation by 35% in primary suspension cultures of mammalian cells, is nearly the same as D0 for survival of clonogenic cells of the same lines and tissues. The extent of DNA synthesis suppression is assessed by impulse 3H-thymidine incorporation in the acid-insoluble fraction of irradiated cells. The values of Di determined in this way for HeLa cells, Ehrlich ascites tumor cells, mouse bone marrow and thymus cells are 2.0, 1.5, 1.5, and 1.0 Gy, respectively; as determined by clonogenic capacity of these cells, Di = 1.9, 2.0, 1.3, and 1.0 Gy, respectively.     

Increase in the number of antibody producing cells during combined cultivation of lymph node cells from immunized animals with intact bone marrow cells]

An increase in the amount of direct and indirect plaque-forming cells in mixed cultures of lymph node cells from mice primed and challenged with sheep red blood cells with syngenic and allogenic bone marrow cells from intact donors was observed. The amount of plaque-forming cells in mixed cultures reached the maximum level in 9--11 hours of cultivation and then fell to the initial level. The authors believe that in mixed cultures of lymph node cells from immune mice and bone marrow cells from intact donors there occurs an involvement into the antibody synthesis of new cells of one of the two cell populations cultured.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

Differential heat shock response of primary human cell cultures and established cell lines

The influence of hyperthermia on the cellular growth and protein synthesis pattern from primary human brain tumour cells and skin fibroblasts was compared with established and experimentally transformed tumour cell lines. Primary cell cultures did not show any visible morphological changes after 42 degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment.     

Modulations in mouse hemopoiesis after engraftment with Lewis lung (3LL) carcinoma cells

Hemopoietic changes in male C57BL/6Cum BR mice engrafted with Lewis lung carcinoma (3LL) were evaluated between day 7, when palpable tumors were present, to day 30 postengraftment. All experimental animals demonstrated decreasing hematocrits (down 40% by day 30) with concurrent leukocytosis which by day 30 postengraftment had reached levels 13.4 times normal. The myelocytic/erythrocytic ratio for normal animals was 1:3 (bone marrow: spleen). The ratio for engrafted animals ranged between 10:1 and 40:1. This apparent shift in production priorities is even more significant in light of the fact that femoral bone marrow cellularity had decreased by 33% on day 17. Splenomegaly, evident by day 7, was seven times control by day 17. Clonogenic analysis of erythroprogenitor cell concentrations revealed an inverse relationship between bone marrow and spleen. 27 days after engraftment, splenic populations demonstrated significant increases in colony forming unit-erythroid (115-fold), burst forming unit-erythroid (7.4-fold), whereas bone marrow concentrations had decreased (6-fold). This report suggests that initiation of 3LL tumor in mice results in a change in the degree of hematopoietic priorities and participation of erythroid organs.     

Protective effect of N-stearoylethanolamine in suspension and in nanocomposite complex in the organs of mice with the Lewis carcinoma under doxorubicin intoxication administration

The antioxidant effects of N-stearoylethanolamine (NSE) in the nanocomplex composition and in suspension are shown on the model of intoxication by doxorubicin in conditions of development of the Lewis carcinoma in the heart, kidneys and liver tissue and in the blood plasma of female mice. The NSE suspension reduces the level of urea in the blood plasma of mice with the Lewis carcinoma, which growth was revealed as a result of introduction of doxorubicin. Under introduction of nanocomplex the amount of urea remains at the level of that in the intact mice. In the blood plasma of mice with the Lewis carcinoma the NSE suspension and nanocomplex reduce activity of aspartate aminotransferase, the basic marker of necrosis of the heart tissue, growth of which was caused by the tumour development. Doxorubicinum increases activity of alanine aminotransferase, the marker of the liver lesion; introduction of NSE in the nanocomplex composition prevents the growth of the enzyme activity. N-stearoylethanolamine, both in the nanocomplex and in suspension, modulates activity of enzymes of antioxidantive protection of the heart, kidney and liver tissue of mice with the Lewis carcinoma.     

Adriamycin-induced centric fusions in mouse chromosomes: in vivo and in vitro analysis

The nature of metacentric chromosomes induced by the anthracycline antibiotic adriamycin (ADR) in mice was studied. The analysis of bone marrow cells of mice injected intraperitoneally with a single dose of the chemical and sacrificed at different posttreatment lapses demonstrated the persistence of biarmed chromosomes even after 30 days. Observations of BUdR-substituted chromosomes from primary cell cultures after one replication cycle revealed the maintenance of DNA polarity through the centromere in most of the metacentric chromosomes. These facts could be considered as a good indication that ADR induces Robert-sonian fusions in mouse chromosomes.     

Nature of the target cells in bone marrow B-suppressor action studied on a simplified model of suppression activity registration

The addition of bone marrow cells to spleen cells and lymph node cells stimulates by mitogents, but not to fibroblast-like cells, leads to a significant reduction of DNA synthesis in mixed cultures in vitro. The suppression effects appears only in two days and the suppressor cell activity is the stronger, the intensive is the target cell proliferation. It is shown that intact bone marrow cells can suppress the lipopolysaccharide-activated bone marrow cell proliferation in vitro. A conclusion may be draw that cells of the lymphoid system serve as target cells for the bone marrow suppressor cells, and the role of these lymphoid system cells is to control immunogenesis processes by suppressing the target cell proliferation activity in the bone marrow.