Triggering of T-cell leukemia and dissemination of T-cell lymphoma in MMP-9-deficient mice.

Previous studies have shown that high levels of MMP-9 can be detected in the serum of patients with various lymphoid malignancies and in leukemia/lymphoma culture supernatants. Indeed, aggressive forms of lymphoma constitutively produce MMP-9 and its elevated levels in the serum or in tissues correlate with advanced stage and poor patient survival. In vitro, MMP-9, which is also produced by the host peritumoral cells in response to the presence of tumors, plays an important role in migration of tumor cells through artificial basement membranes or endothelial cells. In this study, using MMP-9-deficient mice, we show that absence of MMP-9 does not prevent the development of primary T-cell leukemia. Furthermore, MMP-9-deficient cell lines retained their tumorigenic potential, as shown by their ability to induce thymic lymphoma in young syngeneic wild-type animals. In addition, these MMP-9-deficient tumor cells disseminate in normal mice, or mice that are deficient for MMP-9, indicating that tumor growth and dissemination can occur in total absence of MMP-9. These results show for the first time than lymphoma growth can occur in total absence of MMP-9 and have consequences for therapy of invasive cancers with inhibitors of MMPs.

Absence of negative growth regulation in three new murine radiation-induced myeloid leukemia cell lines with deletion of chromosome 2.

Murine radiation-induced acute myeloid leukemia (RI-AML) may be considered as the experimental counterpart of human secondary leukemia. Three new myelomonocytic cell lines derived from RI-AML and carrying a partially deleted chromosome 2 are described. The RI-AML cells responded with increased proliferation after being incubated with the hemopoietic growth factors rG-CSF, rGM-CSF and IL-3. Increased proliferation of the same extent without any effect in differentiation, was also demonstrated in the RI-AML cells after incubation with IL-6 and with mouse lung conditioned medium (CM) and Krebs ascites tumor cells CM which induce differentiation in normal and most leukemic myeloid cells. Down-regulation of the c-myc gene and induction of (2'-5') oligo-adenylate synthetase (reflecting autocrine interferon secretion), two essential mechanisms operating during arrest of growth and concomitant differentiation, were demonstrated to be absent in RI-AML cells. In contrast, the M1 cells responded to the above differentiating factors with growth arrest and differentiation and with appropriate c-myc down-regulation and synthetase induction. The genetic basis for the distinct RI-AML cells' behavior may be connected with the loss or structural and/or functional abnormalities of DNA sequences located in the deleted part of chromosome 2 or in the respective allele. The presently described new RI-AML cell lines may be used for studies concerning myeloid leukemogenesis in general and secondary leukemia in particular.

Establishment and characterization of a transplantable erythroblastic leukemia in C3H mice.

A disease with the characteristics of an erythroblastic leukemia was induced by X-ray irradiation of 300 Rads in C3H mice. The leukemia is transplantable in syngeneic mice by i.v. injection of the spleen cells. The mice show almost pure erythroid cells of various differentiation stages in peripheral blood. The number of total nucleated cells in the peripheral blood increased, but hematocrit and platelet number decreased. Reverse transcriptase activities were measured in spleen and liver of the mice and the data suggested that the leukemia was not induced by retrovirus infection. This leukemia is distinguishable, in this respect, from diseases reported by Friend or Rauscher. The leukemia will offer a good animal model for the studies on non-viral leukemogenesis and disorders of erythropoiesis.

Properties of Na+/K+ ATPase and alkaline phosphatase alter during spontaneous and radiation-induced leukemogenesis in mice.

We characterized properties of Na+/K+ ATPase and alkaline phosphatase in thymocytes or thymoblasts from mice of two strains: AKR, in which thymoma developed spontaneously and C57Bl, in which the development was induced by X-irradiation (total dose: 5.4 Gy in 3 fractions). We found that before thymoma could be discerned morphologically--properties of the two enzymes altered. There was a decrease in 86Rb uptake and in the rate of ATP hydrolysis per cell (both strains) as well as an increase in alkaline phosphatase activity per cell (C57Bl mice). In both spontaneous and radiation-induced thymomas 86Rb uptake, ATP hydrolysis and 3H-ouabain binding per cell were higher than in normal thymuses. Likewise, alkaline phosphatase activity per cell was higher in thymomas than in thymuses; this increase was accompanied by the appearance of additional isoenzyme(s) (1 in AKR, 2 in C57Bl). These changes were compared with cAMP content and 3H-thymidine incorporation, taken as indicators of the proliferative activity, and their high correlation in both AKR and C57Bl mice allowed to distinguish a pre-leukemic period. In that period thymoblasts clearly differed from the normal ones in Na+/K+ ATPase and alkaline phosphatase properties, as well as proliferation, although the morphology of the thymus was still unchanged.

Terminal deoxynucleotidyl transferase (TdT) in RadLV-induced C57BL thymomas.

Newborn C57BL mice exposed to RadLV at birth were followed and periodically sampled for thymus and bone marrow TdT activity. Tumors detected at or before 100 days had mostly low TdT levels. Large thymic lymphocytic tumors detected after 100 days had elevated total TdT content per gland. Serial samples showed a changing TdT pattern with time. The mid period shifted from low levels to normal-to-high levels for both TdT content and total TdT per gland. No unusual bone marrow TdT activity change preceded the appearance of thymomas. TdT marks a unique precursor cell highly sensitive to RadLV associated with lymphomagenesis. RadLV induced both TdT+ and TdT- thymic lymphomas and both types were found to occur after lymphoma initiation by injection of RadLV into newborns.

Radiation leukemia in C57BL/6 mice. III. Correlation of altered expression of terminal deoxynucleotidyl transferase to induction of leukemia.

The expression of terminal deoxynucleotidyl transferase (TdT) in the thymus and bone marrow of irradiated mice has been examined. Mice given the leukemogenic regimen of irradiation of four weekly doses of 175 rads starting at 1 mo of age show a long-term elimination of TdT activity in the bone marrow and a reduction of TdT activity in thymocytes. In such mice, the reappearance of normal levels of TdT in the thymus appears to only be associated with the onset of overt leukemia. This effect on TdT expression was shown to be uniquely associated with the leukemogenic regimen of irradiation in that nonleukemogenic irradiation or variations such as bone marrow reconstitution or age which reduce leukemias did not show the same phenotypic effects on TdT expression. The basis for the loss of TdT-positive cells was shown not to be due to the lack of the requisite factors involved in differentiation, but rather to the ability of leukemogenic doses of irradiation to reduce or eliminate an inducible bone marrow stem cell. These results are discussed with respect to the possible mechanisms involved in radiation-induced leukemias in mice.

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.

Strain-, age-, and tumor-dependent distribution of terminal deoxynucleotidyl transferase in thymocytes of mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peak I and peak II in thymuses and peripheral lymphoid tissues was examined in a variety of mouse strains in relation to tumor burden and age. TdT peak I was found to be present in all strains at comparable levels, which did not change with age. TdT peak II levels were also comparable for the strains examined at 1 week of age. In contrast to peak I, however, in NIH Swiss and AKR mice, peak II activity decreased rapidly at 2 weeks of age and by 6 weeks of age less than 2% of the initial activity remained. In C57BL/6 mice there was a similar loss of peak II activity with age although this change started at about 4 months of age and by 8 months of age approximately 15% of the initial activity remained. These changes did not appear to be due to the presence of an inhibitor. Leukemic C57BL/6 and AKR mice were also examined for TdT. Both strains characteristically had TdT peak I in peripheral lymphoid tissues infiltrated with transformed thymocytes. AKR mice had only TdT peak I in the thymus, whereas C57BL/6 thymuses had both peak I and II at levels comparable to age-matched controls. No aberrant distribution of TdT was observed in a spontaneous reticulum cell sarcoma or Rauscher MuLV-induced erythroblastosis.