Neoplastic and nonneoplastic lesions in aging mice of unique and common inbred strains contribution to modeling of human neoplastic diseases.

The evaluation of spontaneous lesions in classical inbred strains of mice has become increasingly important because genetically engineered mice (GEMs) are created on these backgrounds. Novel inbred strains-genetically diverse from classic strains-are valuable both as a new background for GEM mice and to increase the genetic variation found in laboratory mice. Newly arising spontaneous genetic alterations in commonly used strains may also lead to new and valuable mouse models of disease. This report evaluates gross and histological lesions in relatively new, classic, and rarely explored mouse inbred strains. Pathological lesions of 1273 mice from 12 inbred strains (129S1/SvW, A.CA-H2(f) /W, AKR/W, BALB/cW, BN/aW, C57BL/6 W, C57BL/10 W, C3H/W, C3H (wad) /W, CBA/W, DBA/2 W, and WOM/W) are reported. BN/aW, WOM/W, and C3H (wad) /W are novel inbred strains produced and maintained in the Department of Genetics and Laboratory Animal Breeding at the Center of Oncology, Warsaw, Poland. Both neoplastic and nonneoplastic lesions were examined. The prevalence of lung neoplasms was significantly higher in A.CA-H2(f) /W (33.3%) and BALB/cW (33.8%) mice (P < .01). The prevalence of liver neoplasms was significantly higher in the CBA/W strain (P < .01). Mammary gland neoplasms arose at a greater frequency in C3H/W mice (P < .01). The occurrence of uterine neoplasms was higher in DBA/W and 129S1/SvW mice. AKR/W and WOM/W mice developed T-cell lymphoblastic lymphoma with high frequency (110/121 [90.9%] and 159/175 [90.9%], respectively) before 1 year of age. The occurrence of nonneoplastic lesions in the kidneys of BN/aW mice was increased (P < .01).

The effect of undecanones and their derivatives on tumor angiogenesis and VEGF content.

The in vivo effects of some derivatives of aliphatic ketones (2-undecanone, 3-undecanone, 4-undecanone and their derivatives) on L-1 sarcoma tumor angiogenesis and VEGF content were studied in Balb/c mice. Mice that inhaled 10% solution of 3-undecanone(3-on) or 1% solution of 2-undecanone propylene acetal (Acpr2) for 3 days after tumor cells implantation, presented lower neovascular response measured by tumor-induced cutaneous angiogenesis test (TIA) and lower tumor VEGF content in 5-days tumors, than non-inhaled controls. Other substances presented various effects on tumor VEGF concentration and angiogenesis. Histological examination of lesions collected from mice inhaled Acpr2, or non-inhaled controls, revealed small diffused areas of necrosis in the former group. In both groups, slight to moderate inflammatory infiltrations were seen at the tumor's margin. In Acpr2 group, there were less small blood vessels at tumor's margin than in the control group.

Inhibitory effect of Greenland shark liver oil combined with squalen and arctic birch ashes on angiogenesis and L-1 sarcoma growth in Balb/c mice.

Sharks have been claimed to be resistant to cancer and oil from their livers have been used in Scandinavian folk medicine as anti-tumor drug. Shark liver oil contains 40% or more of squalene. Fish liver oil is also rich in squalene and polyunsaturated n-3 fatty acids. The aim of this work was to determine the anti-angiogenic and anti-tumor effects of these substances, together with another Scandinavian traditional remedy--arctic birch ashes--in Balb/c mice after transplantation of syngeneic L-1 sarcoma. All substances tested, alone or in combinations, significantly diminished cutaneous angiogenesis induced by tumor cells, and tumor growth.

Do altering in ornithine decarboxylase activity and gene expression contribute to antiproliferative properties of COX inhibitors?

Two isoforms of cyclooxygenase (COX) participate in growth control; COX-1 is constitutively expressed in most cells, and COX-2 is an inducible enzyme in response to cellular stimuli. An induction of COX-2 found in neoplastic tissues results in increased cell growth, inhibition of apoptosis, activation of angiogenesis, and decreased immune responsiveness. Although both COX-1 and COX-2 inhibitors are suppressors of cell proliferation and appear to be chemopreventive agents for tumorigenesis, the molecular mechanisms mediating antiproliferative effect of COX inhibitors are still not well defined. This study contrasts and compares the effects of aspirin and celecoxib, inhibitors of COX-1 and COX-2, in rat hepatoma HTC-IR cells. The following were assessed: cell proliferation and apoptosis, ornithine decarboxylase (ODC) activity, and pattern expression of three immediate-early genes, c-myc, Egr-1, and c-fos. We have shown that the treatment of hepatocytes in vitro with the selective COX-2 inhibitor, celecoxib, was associated with induction of apoptosis and complete inhibition of cellular proliferation. Aspirin exhibited a small antiproliferative effect that was not associated with apoptosis. Treatment with celecoxib produced dose- and time-dependent decrease in ODC activity. In addition, at higher drug concentration the decrease in ODC activity was greater in proliferating than in resting cells. Much lesser inhibitory effect on ODC activity was observed in aspirin-treated cells. The two COX inhibitors did not change c-myc expression, significantly decreased the expression of Egr-1, and differentially altered expression of c-fos; aspirin did not change, but celecoxib dramatically decreased the levels of c-fos-mRNA. Our study revealed that celecoxib and aspirin share the ability to inhibit ODC activity and alter the pattern of immediate-early gene expression. It seems that some of the observed effects are likely to be related to COX-independent pathways. The precise mechanisms of action of COX inhibitors should be defined before using these drugs for cancer chemopreventive therapy.

Recovery of dendritic cell counts and function in peripheral blood of cancer patients after chemotherapy.

Dendritic cell (DC) counts and function were assayed in peripheral blood of lymphoma and solid tumor patients before and after chemotherapy. The DC counts declined significantly within the first week from the start of chemotherapy, recovered in the second week, and exceeded the baseline values in the third week. DC recovery was usually similar after the first and after the last cycle of chemotherapy. DC1 and DC2 subsets followed the pattern of reconstitution found for the DC population as a whole. Monocytes and granulocytes recovered 1-2 weeks later than DC. The primary proliferative response to keyhole lympet hemocyanin (KLH), totally DC-dependent, declined within the first week from the start of chemotherapy, and in the majority of patients (including those initially unresponsive) recovered along with DC counts. The recovered responsiveness to KLH, but not to anti-CD3 antibody, disappeared at the end of chemotherapy in lymphoma and some solid tumor patients. Prolonged depletion of CD4+ T cells could contribute to the loss of responsiveness in lymphoma patients receiving multiple cycles of chemotherapy. However, in some solid tumor patients, the reactivity to KLH was absent, despite the reconstitution of both DC and CD4+ T-cell counts. Our data show that numerical reconstitution of DC is not necessarily accompanied by functional recovery. The early recovery of DC should be considered while designing protocols for DC collection for immunotherapy.

A method for directly determining the number of dendritic cells and for evaluation of their function in small amounts of human peripheral blood.

Bone marrow-derived dendritic cells (DC) are highly potent antigen-presenting cells (APC) capable of initiating primary responses of naive T lymphocytes to antigen. Studies on DC in disease have been impeded by the lack of a defined method for accurate DC counting and for evaluation of their function in a small amount of blood. In order to detect and enumerate DC in whole peripheral blood preparations, we applied a direct two-color immunofluorescence method. Blood from healthy donors was stained with a mixture of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) recognizing lineage-associated molecules (CD3, CD14, CD16, CD20, CD57) and phycoerythrin (PE)-conjugated anti-HLA-DR mAb. DC were identified as lineage marker negative (lin-), HLA-DR highly positive cells. The mean percentage of these cells present in peripheral blood leukocytes (PBL) was 0.54%, and the mean absolute DC count was 31.4 x 10(6)/l of blood. DC stained directly in whole blood were heterogeneous with regard to their expression of CD2 and CD4 molecules, and did not express CD80 and CD83 molecules. Expression of CD80 and CD83 on DC was induced following a multistep isolation procedure, including overnight culture. We demonstrated a significant primary proliferative response to keyhole limpet hemocyanin (KLH) in cultures of peripheral blood mononuclear cells (PBMNC). Since primary proliferative response to neoantigens is entirely dependent on DC as APC, the cultures of unseparated PBMNC stimulated with KLH can be used to evaluate DC function in a relatively simple test. This test does not require previous isolation of DC and T lymphocytes and, therefore, can be performed on a small amount of blood. The elaborated flow cytometric method of DC counting in blood and the proliferative test of DC-dependent primary response to neoantigen are currently being applied in an ongoing study on the effect of chemotherapy on DC number and function in cancer patients.

Detection of circulating melanoma cells in peripheral blood by a two-marker RT-PCR assay.

The aim of this study was to develop a highly sensitive two-marker assay for the detection of circulating melanoma cells in patients' blood using a reverse transcriptase-polymerase chain reaction (RT-PCR). We analysed the usefulness of two different sets of markers: tyrosinase and MUC-18 (TYR/MUC-18), and tyrosinase and MART 1 (TYR/MART 1). Total cellular RNA was isolated from 337 blood samples from 80 melanoma patients at different stages of the disease. All patients had undergone primary surgery. Assay sensitivity and specificity were confirmed using three different melanoma cell lines and two different fibroblast lines. In addition, blood from 47 healthy subjects and 10 patients with non-melanoma cancer was used as a negative control. We found that two-marker analysis is more accurate than the single tyrosinase assay. The frequency of melanoma cell detection in patients' blood was about 10% higher when the TYR/MART 1 two-marker assay was used. Using this assay we did not find any statistical correlation between the molecular markers and the UICC stage of disease or the Breslow thickness or Clark level of the primary melanoma. The frequency of melanoma cell detection with the TYR/MUC-18 two-marker assay was even higher than the TYR/MART 1 assay, but unfortunately the MUC-18 transcript was also present in about 20% of healthy subjects. Therefore we do not recommend the use of MUC-18 as a standard value marker.

Inhibitory effect of shark liver oil on cutaneous angiogenesis induced in Balb/c mice by syngeneic sarcoma L-1, human urinary bladder and human kidney tumour cells.

The effect of shark liver oil on cutaneous angiogenesis induced in mice by intradermal grafting of tumour cells was evaluated. It was shown that this substance (Ecomer) suppressed neovascular response in mice grafted with sarcoma L-1 syngeneic cells, human kidney cancer and human urinary bladder cancer cells. In addition, strong stimulatory effect of this drug on mice blood granulocyte number and their metabolic activity was observed.

Genetics of susceptibility to radiation-induced lymphomas, leukemias and lung tumors studied in recombinant congenic strains.

The genetic control of susceptibility to radiation-induced tumors in mice has been tested using the series of 20 CcS/Dem (CcS) Recombinant Congenic Strains, each carrying a different random set of 12.5% of genes of the resistant strain STS/A (STS) on the genetic background of the susceptible strain BALB/cHeA (BALB/c). Two classes of tumors were frequently observed: tumors of the haematopoietic system (lymphomas, myelocytic leukemias) and lung tumors. The results indicate that the genes controlling various aspects of tumor development were segregated in the CcS strain series. Large inter-strain differences were observed in the incidence of lung tumors. With lymphomas and leukemias, we not only observed strain differences in the incidence of tumors and in the latency of their development but also in the type of tumors (T- vs. B-cell lymphomas, myelocytic tumors) and in the frequency of their localized or disseminated (leukemic) form. Surprisingly, the myelocytic tumors, which occur very rarely or not at all in the parental strains BALB/c and STS or in their crosses, developed with high frequency in one of the CcS strains (CcS-2), indicating a unique combination of genes in this strain, which facilitates the development of myelocytic tumors. The effect of these genes is suppressed in the genetic composition of the parental strains. Tests of crosses of the resistant-strain CcS-13 with BALB/c indicated a suggestive linkage of a susceptibility gene for lymphomas to chromosome 5. These tests of the CcS strains illustrate the genetic complexity of the control of radiation-induced tumors in mice and suitability of these model systems to study their different facets.

Surface phenotypes of human T cell lines generated after WGA- and PHA-stimulation.

We have reported earlier that human T lymphocytes from blood or spleens of normal donors can be propagated in long-term culture in the presence of IL-2, after stimulation either with wheat germ agglutinin (WGA) or phytohemagglutinin (PHA) and with the use of repeated restimulation with supernatants collected after 24 h of such cultures. In the present study we demonstrate that lymphoblast lines generated with this method contain predominantly the cells expressing CD3 molecule and alpha beta heterodimer of T cell receptor (TcR alpha beta). In PHA-initiated lymphoblast lines CD4-CD8+ cells prevailed, whereas in lymphoblast lines generated after WGA-stimulation various proportion of CD4+CD8-, CD+CD8- and CD4-CD8- cells were found. In some cell lines a gradual selective outgrowth of TcR alpha beta CD4+CD8- or CD4-CD8- cells was observed. These data indicate that the long-term growth potential of human normal T lymphoblasts is not restricted to a single subset and that the surface phenotypes acquired during differentiation of the cells can be retained even after more than 100 population doublings in culture.

Thymocyte and splenocyte subpopulations in normal and leukemia-bearing mice after adrenalectomy.

Mice with i.p. inoculated leukemia L1210 cells had lower total number of thymocytes, and reduced number of CD4+8+ thymocytes, but increased CD4+8- subpopulation. Adrenalectomy reduced the CD4-8+ subset of thymocytes. Leukemia in adrenalectomized animals induced similar changes in the number of thymocyte subpopulations as observed in normal animals. However, a decrease in CD4+8- thymocytes was noted and the animals had decreased number of CD4-8- cells as well. Splenocytes of CD4+8- subpopulation were more numerous in adrenalectomized animals. Leukemia induced increase in the number of CD4+8- and CD4-8+ splenocytes in both normal and adrenalectomized animals.

Abnormalities in T cell interactions with the extracellular matrix proteins in a patient with Wegener's granulomatosis.

T cell interactions with the extracellular matrix proteins and cultured human endothelium were studied in a patient with Wegener's granulomatosis and other cases of vasculitis. Markedly enhanced costimulation of T-lymphoproliferative responses mediated by collagen and fibronectin were found in patients with severe forms of vasculitis, particularly with necrotizing changes. In addition, enhanced adhesion to collagen IV was found in the Wegener patient. T cell adhesion to resting and inflamed endothelium varied from normal to increased.

Effect of purinergic receptor antagonists suramin and theobromine on tumor-induced angiogenesis in BALB/c mice.

The purinergic receptor antagonists suramin (SRN) and theobromine (TBR) were examined for their anti-angiogenic activity in BALB/c mice. SRN or TBR were subcutaneously administered to BALB/c mice in doses of 1-125 mg/kg body weight on days 0, 1 and 2 after intradermal inoculation of E14/W lung carcinoma cells. It was shown that SRN and TBR inhibited tumor-related angiogenesis. Accordingly, it may be suggested that purinoceptor antagonists may inhibit neovascularization in tumor growth and metastasis.

Induction of ornithine decarboxylase in normal and protein kinase C--depleted human colon carcinoma cells.

To examine the role of protein kinase C (PKC) in induction of human colon adenocarcinoma cell line, DETA/W, by polypeptide growth-promoting factors, ornithine decarboxylase activity (ODC) and DNA synthesis were determined in cells depleted of PKC. PKC depletion was achieved by prolonged cultivation (more than 30 passages) with 10(-6) M phorbol 12-myristate 13-acelate. Lack of PKC in studied cells was proved by measurements of PKC activity and immunoreactivity. Although ODC activities and DNA syntheses in PKC-depleted cells were decreased by about 40-50% compared to normal DETA/W cells, the percentage increase of these mitogen-responsive reactions was quantitatively similar in both cell sublines. These results raise the possibility that not all of the biological responses to growth factors are connected with the activation of calcium-dependent PKC.

Changes of thymocyte subpopulations induced by activities diffusing from leukemia L1210 cells.

Mice with intraperitoneally inoculated leukemia L1210 cells had a lower total number of thymocytes and a markedly reduced percentage of L3T4+ Lyt2+ (CD4+ 8+) thymocytes, but increased subpopulations of L3T4+ Lyt2-, L3T4- Lyt2+, L3T4-Lyt2-. The percentage of Thy 1.2+ thymocytes was unchanged as compared with control animals. A similar effect was observed is suggested that leukemia L1210 cells produce some activities which diffuse through 0.22 microns filters and influence the subpopulations of thymocytes.

Inhibition of calf thymus DNA polymerase alpha and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine.

6-(p-n-Butylanilino)uracil and N2-(p-butylphenyl)guanine inhibited the activity of DNA polymerase alpha from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases beta and gamma) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5'-triphosphate and did not occur in the reaction of DNA polymerase alpha with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases alpha.

IgM antibody-dependent cell-mediated cytotoxicity in the Moloney sarcoma virus system: the involvement of T and B lymphocytes as effector cells.

Antibody-dependent cell-mediated cytotoxicity in the Moloney sarcoma virus (MSV) system was analyzed with respect to the subpopulations of effector cells involved in tumor target cell destruction when IgM was used as the sensitizing antibody. With unfractionated sera from animals that had undergone regression primary MSV tumors it was found that macrophages did not contribute to the cytotoxicity induced by normal spleen cells that were syngeneic to the target cells. The IgM fraction of MSV regressor sera was found to induce cytotoxicity against the target cells by immunoadsorbent column-fractionated normal spleen cells, which were either depleted of T cells or B cells, according to the specificity of the columns. Immune IgM was also found to potentiate the activity of MSV regressor spleen cells that had been similarly fractionated. Furthermore, IgM antibody was found to induced cytotoxicity by normal spleen cells which had been depleted of either T or B cells by the appropriate antiserum (anti-T or anti-Ig) in the presence of complement and subsequent recovery of the viable cells by trysinization, filtration, and washing. However, spleen cells treated with both anti-T and anti-Ig sera simultaneously in the presence of complement and subsequet recovery of viable cells, were not induced to be cytotoxic against the IgM-coated tumor target cells. Further support oy T cells was provided by an experiment showing the induction with IgM of cytotoxicity against the target cells by normal thymocytes.

Antibody-dependent lymphocyte cytotoxicity in the murine sarcoma virus system: activity of IgM and IgG with specificity for MLV determined antigen(s).

Antisera with specificity for Moloney leukemia virus-(MLV) determined antigen(s) were studied for their ability to induce MLV antigen bearing target cell reduction by lymphocytes in microcytotoxicity assays. Sera from animals which had regressed Moloney sarcoma virus (MSV) tumors as well as sera from animals with progressively growing MSV tumors were found to induce normal lymphocytes to be active against the targets. Regressor serum was found also to induce cytotoxicity by immune lymphocytes from a tumor-bearing animal 15 days after MSV and from a regressor 50 days after MSV infection. Both the 19S and 7S Sephadex G-200 fractions of the antisera were found to induce cytotoxicity by normal lymphocytes and to potentiate the cytotoxicity of MSV immune lymphocytes. These activities were shown to be IgM and IgG, respectively, by the use of Sepharose-coupled anti-mouse IgM and anti-mouse IgG columns. All activity was removed by passing sera over both columns.