Transcriptional regulation and biological significance of the insulin like growth factor II gene.

The insulin like growth factors I and II are the most ubiquitous in the mammalian embryo. Moreover they play a pivotal role in the development and growth of tumours. The bioavailability of these growth factors is regulated on a transcriptional as well as on a posttranslational level. The expression of non-signalling receptors as well as binding proteins does further tune the local concentration of IGFs. This paper aims at reviewing how the transcription of the IGF genes is regulated. The biological significance of these control mechanisms will be discussed.

Growth factors and apoptosis in development. The role of insulin like growth factor I and TGFbeta1 in regulating cell growth and cell death in a human teratocarcinoma derived cell line.

The balance between different cell populations in the developing organism is controlled by regulating the rates of multiplication, differentiation or death of its constituent cells. The human teratocarcinoma derived cell line Tera 2, which in several aspects mirrors early embryonic cells, can be induced to undergo programmed cell death (apoptosis) by depriving cell cultures of serum. This study demonstrates that this process can be reversed by replacing serum with physiological concentrations of insulin like growth factor I (IGF I). As a result, IGF I enhances the rate of Tera 2 cell proliferation in serum free medium. In contrast, Transforming Growth Factor beta1 did not exert any effect on growth or apoptosis in Tera 2 cells. The results indicate that one effect of growth factors on pluripotential cells is to regulate the balance between cell proliferation and cell death.

Insulin-like growth factor II prevents apoptosis in a human teratoma derived cell line.

Aim-To study how insulin-like growth factor II (IGF-II) affects the behaviour of human teratoma cells.Methods-The human pluripotential teratoma cell line Tera 2 was cultured under serum-free conditions in the presence or absence of IGF-II. Effects on cell proliferation and apoptosis as well as on the expression of the proto-oncogene c-myc were studied.Results-In this study we show that Tera 2 cells deprived of serum undergo programmed cell death (apoptosis). The onset of nuclear fragmentation was observed 12 hours after serum withdrawal. The morphological changes of the Tera 2 cell nuclei were confirmed by the occurrence of a nucleosome ladder. However, the constitutive expression of the proto-oncogene c-myc was not decreased in parallel with initiation of apoptosis. The apoptotic response to serum withdrawal could be counteracted by simultaneous addition of IGF-II. In addition it was found that human testicular tumours (seminoma and embryonal carcinoma) contain raised levels of insulin-like growth factors.Conclusions-The precise roles of IGF-I and IGF-II have been unclear, and there is overwhelming evidence against these factors as primarily transforming agents. The finding that IGF-II apparently counteracts apoptosis in vitro may well explain its effects on tumours in vivo.

Comparative two-stage cancer tests of ethylene oxide, N-(2-hydroxyethyl)-N-nitrosourea and X-rays.

Mutagenicity tests have shown that the potencies of ethylene oxide and other alkylating agents relative to that of low-LET ionising radiation are approximately the same in different biological systems. In the present study this relationship, the radiation-dose equivalence ("rad-equivalence") of doses of genotoxic chemicals, was tested for the induction of tumours in skin and lung of mice using different initiation-promotion protocols. The initiators used were X-rays, ethylene oxide and N-(2-hydroxyethyl)-N-nitrosourea (HOENU). This short-term treatment was followed by treatment with the "promoters" 12-O-tetradecanoylphorbol 13-acetate (TPA) and carbon tetrachloride. Unexpectedly, the animals treated with carbon tetrachloride did not show treatment-related liver tumours, but exhibited precocious death, mostly with lung tumours. According to estimates from in vitro tests the total in vivo dose from exposure to 400 ppm for 4 x 5 h corresponds to 700 rad-equivalents. Although still with great statistical uncertainty, this ratio is supported by the observed time-dependent frequencies of animals with papillomas (in the TPA series) and lung tumours (in the carbon tetrachloride series). Animals treated with HOENU exhibited high incidences of tumours of both these types in approximate agreement with the higher rad-equivalence of the treatments with this compound.

The influence of selenium, vitamin E, and oestrogen on the development of tumours in mice exposed to 90Sr.

The primary object of this experiment was to evaluate the potential role of the antioxidants, selenium and vitamin E, in the anti-tumour defence of mice internally irradiated with 90Sr. Comparison in terms of neoplastic response was made between mice kept on a selenium and vitamin E deficient diet and mice given the same deficient diet but administered selenium and/or vitamin E in a controlled manner. The influence of simultaneous oestrogen treatment, known to promote radiogenic osteosarcoma induction, was also investigated. Non-irradiated mice were used as controls. Results are presented as crude and actuarial tumour incidence. No significant difference in tumour yield or actuarial tumour incidence was found when the differently treated mouse groups were compared, and accordingly no support was gained for the theory that the antioxidants selenium and vitamin E constitute a critical part of the complex defence system against neoplasms.

The effects of leukemia inhibitory factor (lif) on cell multiplication and locomotion in human teratocarcinoma cells.

The human teratocarcinoma cell line (Tera 2) could be stimulated to a moderate increase in cell number in serum-free medium by addition of 5 ng leukemia inhibitory factor (LIF)/ml. However this effect was only observed in short term (24 h) cultures. By comparing cell numbers with thymidine incorporation data and proportion intact cell nuclei, we concluded that this short term increase in cell number was due to enhanced cell survival rather than a real increase in the proportion of cells traversing the cell cycle. When increased concentrations of LIF were added a preferential effect on clonal cell locomotion was observed. 50-200 ng of LIF stimulated cell movement but exerted no effect on Tera 2 cell proliferation at any time interval studied.

Effects of glucan on the reticuloendothelial system and on the development of tumors in 90Sr-exposed mice.

A series of experiments was conducted to examine the effect of glucan on the reticuloendothelial system (RES) and on the development of 90Sr-induced osteosarcomas and malignant lymphomas in CBA/S mice. Glucan demonstrated a strong RES-stimulating effect, as evidenced by a dose-related increase in lysozyme levels in the plasma and an enlargement of the liver and spleen. Weekly injections of glucan between 150 and 250 days after exposure to 90Sr suppressed the actuarial appearance of the fibroblastic type of osteosarcomas and stimulated the emergence of malignant lymphomas. Glucan itself had no tumorigenic effect in mice not exposed to 90Sr.

Impact of 90Sr on mouse natural killer cells and their regulation by alpha interferon and interleukin 2.

Male CBA/SU mice were exposed to ionizing radiation by intraperitoneal injection of the bone-seeking beta-emitter 90Sr. NK-cell lytic activities in spleen, peripheral blood, and lymph nodes were severely depressed or completely abolished. In contrast, production of the NK regulatory proteins alpha interferon (IFN-alpha) and interleukin 2 (IL-2) was normal 5-8 weeks after 90Sr injection. IFN-alpha, produced in vivo or in vitro by cells from injected mice, was able to enhance strongly NK lytic activities. These data indicate that 90Sr acts on the bone marrow, where it interferes with the maturation and seeding of NK precursor cells. The mechanisms regulating NK activities in peripheral organs remained relatively unchanged. Finally, we did not detect any major organ redistribution of NK cells as a result of 90Sr irradiation.

Immune competence in 90Sr-exposed, adult thymectomized and antilymphocyteglobulin-treated CBA mice. II. Reticuloendothelial phagocytic function and in vitro mitogen responsiveness of spleen cells.

The hypothesis that immune system failure plays a role in the development of radiation induced tumours was recently investigated experimentally. Young adult CBA mice, intact or immunocompromised by adult thymectomy (ATx) and/or anti-lymphocyteglobulin (ALG) treatment, were exposed to single doses of 90Sr, after which tumour development was monitored. To evaluate the experimental results required knowledge about the immunological responsiveness of the mice. The present paper contributes to that knowledge by reporting on the in vitro responsiveness of lymphoid cells to mitogens (LPS, PHA, Con-A) and the in vivo phagocytic functioning of the reticuloendothelial system (RES), measured as the rate of clearance of 125I-albumin micro-aggregates in peripheral blood. 90Sr, ATx and ALG-treatments, separately and in combination, suppressed mitogenic lymphoid cell activation, whereas the RES phagocytic function remained unchanged, except in response to 90Sr + ALG treatment, which seemed to slightly inhibit phagocytic activity.

Radiostrontium-induced oncogenesis and the role of immunosuppression. II. Influence of 90Sr dose, adult thymectomy and antilymphocyteglobulin treatment on the development of lympho-reticular and extraskeletal, neoplastic lesions in CBA mice.

The significance of depressed immune function for the development and progression of tumours induced by 90Sr (mainly osteosarcomas and malignant lymphomas) was investigated in a series of experiments by comparing the tumour responses in normal mice with those in immunocompromised mice. The present paper (part II) reports on lympho-reticular (LR) and extraskeletal neoplastic lesions in male CBA/SU mice after exposure to different single doses of 90Sr with or without additional immunosuppression by adult thymectomy (ATx) and/or prolonged antilymphocyteglobulin (ALG) treatment. Neoplastic lesions in bone were reported in part I. The status of the animal's immune system and responsive ability were examined in parallel experiments. The tumour yields were analysed in relation to the dosage of 90Sr and the immunosuppressive treatments employed. Although the incidences and latency times of induced tumours were clearly dose-dependent, they were never significantly influenced by ATx/ALG treatments. Thus, no substantial support was gained for the theory that the immune system plays a controlling or modifying role in 90Sr carcinogenesis. The results, which are in agreement with the bone tumour responses, suggest that 90Sr induced tumours either do not express the antigens necessary for immune rejection or that the decline in immune responsiveness induced by ATx/ALG was of little consequence for tumour development and spread. The pathogenesis of 90Sr induced malignant lymphomas (MLs) and their immunophenotypes are discussed.

Immune competence in 90Sr-exposed, adult thymectomized and antilymphocyteglobulin-treated CBA mice. I. Allogenic skin graft reaction.

CBA mice subjected to either adult thymectomy, internal exposure to 90Sr or antilymphocyteglobulin treatment separately, or to combinations of the three were tested for cellular immune competence using their reaction to allogenic skin grafts. Peripheral blood white cell counts did not reveal any obvious correlation between the degree of mononuclear cell depletion and the ability to accept grafts, suggesting that the particular treatments depleted specific fractions of mononuclear cells, differing in their extent of involvement in the rejection process. No single treatment alone induced a significant prolongation in the time elapsed before graft rejection. Adult thymectomy followed by appropriate antilymphocyteglobulin treatment induced severe lymphocytopenia and a profound suppression of the cell-mediated immune system, as evidenced by the acceptance of allogenic skin grafts. When applied to 90Sr-preexposed mice the same treatment induced lifelong acceptance of grafts, indicating a similar, though weaker immunosuppressive impact of 90Sr. Hence it was possible to significantly enhance immunosuppression in 90Sr-exposed mice. This in vivo model should be useful when investigating the role of immunological responsiveness in radiation carcinogenesis.

Radiostrontium-induced oncogenesis and the role of immunosuppression. I. Influence of 90Sr dose, adult thymectomy and antilymphocyteglobulin treatment on the development of neoplastic and preneoplastic lesions in the skeleton of CBA mice.

Ionizing irradiation by incorporated strontium-90 exerts two major effects: it induces tumours (mainly osteosarcomas and lymphoreticular tumours) and depresses the immune system. The interrelation between these functions, i.e. the significance of decreased immunological responsiveness in the oncogenic process, remains unclear. The influence of the 90Sr dose and the role of immune modulation on the tumour yield, were investigated in young adult CBA mice. The animals were exposed to different single doses of 90Sr and, in addition, some groups were subjected to long-term unspecific immune suppression by adult thymectomy (ATx) and/or prolonged antilymphocyteglobulin (ALG) treatment. The present paper (part I) reports on the effects of the treatments on bone tumour responses as reflected by incidence, multiplicity, latency time, histologic characteristics and growth behaviour. The histogenesis of osteosarcomas, as evidenced morphologically by preneoplastic and early neoplastic growth, is illustrated and discussed. The results demonstrate a positive dose-response relationship for osteosarcomas, in which the relative incidences of the various osteosarcoma subtypes were differentially affected. Thus, well-differentiated tumours were gradually replaced by less differentiated types as the dose decreased. A correlation was also observed between the incidence of osteosarcomas and that of assumed preneoplastic lesions in the same bones and sites. Immune suppression by ATx and/or ALG did not distinctly alter the neoplastic or preneoplastic responses at any dose-level of 90Sr.

Influence of 90Sr, adult thymectomy and antilymphocyteglobulin on haematopoietic tissues and peripheral blood leucocytes in CBA mice.

The role of long-time immune suppression in carcinogenesis induced by the long-lived internal emitter 90Sr, is investigated in an ongoing study. The experimental design is based on the assumption that impaired immune responsiveness, by other means than 90Sr, might increase the neoplastic response in exposed individuals, and thus reflect a protective function, if existing. Intercomparison is made of the tumour yield in mice exposed to different single doses of 90Sr and simultaneously subjected or not to long-term immune suppression by adult thymectomy (ATx) and/or antilymphocyteglobulin (ALG) treatment. Information on the general condition and responsiveness of the immune system, in the respective models, during tumour expectancy time, is essential for a conclusive evaluation of the results. To meet these demands the present paper reports on histopathologic alterations in immune organs and changes in white blood cell counts, induced by the different combinations of 90Sr, ATx and ALG treatment. The results confirm the prediction, that ATx + ALG is an efficient and, with respect to the purpose of the study, suitable treatment for additive long-term depression of the immune system in 90Sr irradiated mice, evidenced in particular by increased depletion of monomorphonuclear cells (MNC) in lymphoid organs and peripheral blood. Subsequent reports will deal with functional immune parameters.

Influence of 90Sr, adult thymectomy and antilymphocyte globulin on T-cells in mouse peripheral blood.

Three groups of male CBA mice were treated by (1) 90Sr injection (14.8 kBq/g body weight i.p.), (2) adult thymectomy (ATx) + antilymphocyte globulin (ALG), and (3) 90Sr + ATx + ALG, respectively, and one untreated group served as a control. The relative number of T-lymphocytes was determined in peripheral blood mononuclear cells of individual animals using a dye exclusion cytotoxicity assay with antisera against the T-cell surface membrane marker Thy 1.2 and complement. Total and differential leucocyte counts were also performed. 90Sr irradiation decreased the total number of leucocytes irrespective of type, and the combined treatment of ATx and ALG decreased mainly mononuclear cells and particularly T-cells. The most advanced T-cell depletion in peripheral blood was found in the 90Sr + ATx + ALG treated group with a 97 per cent reduction as compared with untreated controls. ATx + ALG thus proved to be useful for blood T-cell depletion in mice treated simultaneously with 90Sr, and might provide a valuable tool in investigations on the possible role of cell-mediated immune response in radiation-induced oncogenesis, with particular emphasis on selective depletion within the monomorphonuclear compartment.

Age and dose related carcinogenicity of 90Sr.

90Sr was given at three different dose levels (29.6, 14.8 and 7.4 kBq/g b.w.) to groups of mice aged 300, 150, 75 and 25 days. It was found that the incidence of osteosarcomas was highest in the 75-day-old-group and lowest in the two oldest age groups. The frequency of lymphoreticular tumours was inversely dose-related (highest incidence in the lower dose series) and not dependent on age at the time of 90Sr injection. The frequencies of soft tissue tumours indicate that these tumours are more related to age than to the dose employed.

Induction of neoplasia by 140Ba in mice.

C57Bl mice of both sexes were given intraperitoneal injections of 140Ba-nitrate at 3 different dose levels (55.5, 37.0 and 18.5 kBq/g b.w.). Osteosarcomas were found in all groups and the osteoblastic type was the most common. In the middle and the highest dose groups, female mice developed tumours more frequently than the males. The irradiation doses were calculated to the femur, lumbar vertebrae and sternum and turned out to vary between 6 and 0.7 Gy.

Effect of syngeneic bone marrow and thymus cell transplantation to 90Sr irradiated mice.

Mice irradiated with 90Sr in doses of 14.8 and 25.9 kB/q/g body weight were given bone marrow or thymic cells intravenously at monthly intervals, or in single doses at 120 or 170 days after injection of 90Sr. At the low dose level a high incidence of lymphoreticular tumours in all cell treated groups occurred as compared with animals irradiated with 90Sr only. At the high dose level only the bone marrow transplanted group contracted a high incidence of lymphoreticular neoplasia. Furthermore, a somewhat decreased osteosarcoma incidence in the cell transplanted animals appeared to be indicated. However, the results obtained are inconsistent and difficult to evaluate. Therefore, it seems necessary to repeat the experiments, if more precise conclusions should be possible to draw.

Induction of pituitary tumours by combination of oestrogenic hormones and 90Sr.

The present investigation was initiated to analyse the carcinogenic effect of combined treatment with 90Sr and oestrogenic hormones or corticosteroids in inbred CBA mice. Pituitary tumours appeared in a remarkably high incidence in mice treated with oestrogens + 90Sr in low doses--0.925 kBq/g body weight (44%) and 1.850 kBq/g body weight (37%)--as compared with mice treated with 90Sr only--1 and 3 per cent, respectively. The syncancerogenic effect is ascribed the oestrogen induced proliferation of pituitary cells and their increased sensitivity to radiation. The reverse relation found between number of pituitary tumours and dose of 90Sr in oestrogen treated mice is explained by the reduction of the survival time with increasing dose. Preneoplastic histologic changes of the pituitary are described and pituitary tumours which mainly appeared in pars distalis, are classified according to the growth and tinctorial characteristics.