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The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on the in vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblast-enriched cultures using 3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10(-11) to 10(-7) M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10(-11) M. At nM concentrations, neither DEXA nor DHT stimulated 3H-thymidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10(-7)-10(-9) M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 +/- 115 fmoles/10(5) cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10(5) cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of 3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals. It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.

Correspondencia con motivo de la muerte de Eugenia Sacerdote de Lustig Fil: Dosne de Pasqualini, Christiane. Unidad documental simple

We have demonstrated that medroxyprogesterone acetate (MPA), when administered in high doses, induces mammary carcinomas in virgin female BALB/c mice. Since one of the possible explanations for this effect was its progestagenic effects, we decided to investigate whether progesterone (Pg) alone could also induce mammary adenocarcinomas in our model and if MPA at doses lower than those used to establish the model was also carcinogenic. A total of 136 mice were subdivided into 3 groups: Group 1, 44 mice were implanted s.c. with 40 mg Pg silastic pellets at the beginning of the experiment, and 6 months later with a 20 mg Pg pellet; Group 2, 45 mice were similarly treated with MPA pellets; Group 3, 47 mice were inoculated s.c. with 40 mg MPA every three months. At the end of 20 months, 9 animals had developed mammary tumors in Group 1, 18 in Group 2 and 34 in Group 3 (actuarial incidence = 28%, 58%, and 98%, respectively); tumor latency was similar in all groups: 46.2 +/- 13.1, 51.3 +/- 9.9, and 50.1 +/- 2.1 weeks, respectively. Seven (Group 1), 14 (Group 2), and 25 (Group 3) tumors were transplanted into syngeneic mice to determine progestin dependence. All tumors, except one from Group 1, were histologically characterized. In Group 1 (Pg 60 mg), 4 tumors (67%) were infiltrating lobular carcinomas and 2 were ductal carcinomas (33%). In Group 2 (MPA 60 mg), 2 tumors (14%) were lobular and 12 were ductal adenocarcinomas (86%) (Group 1 vs Group 2: p0.05), whereas in Group 3 (MPA 160 mg), 8 were lobular carcinomas (32%) and 17 were ductal carcinomas (68%). In syngeneic passages all lobular tumors behaved as progestin independent (PI) and ductal tumors as progestin dependent (PD). All ductal tumors, except one, expressed estrogen receptors (ER) and progesterone receptors (PR), whereas receptor expression was variable in lobular carcinomas. It can be concluded that Pg induces mostly lobular, PI mammary tumors in BALB/c female mice. The fact that most MPA-induced tumors are ductal and PD suggests that the two hormones use different carcinogenic pathways.

Cellular proliferation involves different regulatory molecules that include hormones and growth factors. Interestingly, progestins can stimulate as well as inhibit the growth of tumors depending on the experimental design (1). In mammary cancer, polypeptide growth factors, such as EGF, TGFα, TGFβ, and IGF-I, IGF-II, may play either an autocrine or a paracrine role. The present study investigates the presence of these factors and their receptors in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA).

The ability of neonatal murine F1 cells to regulate parental graft vs. host (GvH) reactions was investigated. Neonatal F1 splenocytes were able to decrease significantly the deleterious effects of systemic GvH reactions induced either with maternal or paternal splenocytes in a third party strain. Both neonatal F1 splenocytes or thymocytes were able to decrease local GvH reactions induced with maternal splenocytes towards paternal histocompatibility antigens. In the same experimental conditions, however, neonatal F1 cells were unable to decrease local GvH reactions induced with paternal splenocytes towards maternal histocompatibility antigens; using different numbers of neonatal F1 cells not only was no suppressive effect detected but even, a significant increase in GvH was registered. Similar results were obtained when mortality assays were carried out. It can be concluded that neonatal F1 mice differ in their capacity for regulating parental alloreactive T reactions against self histocompatibility antigens either of maternal or paternal origin.

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increased in vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, the in vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.

In a previous paper we reported early immunoregulatory mechanisms involving not only the appearance of progressive suppression but also significant increases in alloreactive T levels in paraaortic lymph nodes (PALN) and spleen, not only in allogeneic but also in syngeneic pregnancies. Taking into account the hypothesis of the superposition of the alloreactive and the anti-self plus conventional antigens T sets of cells, we investigated whether immunization with conventional antigens was able to alter alloreactive T levels. Weekly i.p. doses of rabbit red bloods cells (RRBC) in BALB/c mice resulted in a dose-dependent increase in spleen alloreactivity as determined by graft-versus-host (GvH) assays in strain combinations differing at H-2 level but not in those sharing the same H-2 with BALB/c. The increases could be significantly decreased by an anti-idiotype anti-RRBC serum. Pretreatment with i.p. weekly doses of sheep red blood cells (SRBC) before mating was able to induce dose-dependent fetal damage when the parents differed at the H-2 level. SRBC immunization in one of the uterine horns induced increases in PALN weight which were much higher in progesterone-pseudopregnant than in virgin mice; T alloreactivity was significantly increased in the draining PALN only in pseudopregnant females. These results favour the postulation of the superposition between the alloreactive and the anti-self plus conventional antigens T sets of cells and suggest a possible role for conventional fetal antigens (non H-2) in triggering immunoregulatory mechanisms operating in pregnancy.

The sponge matrix allograft model made possible the growth of an AKR lymphoma in a certain percentage of BALB/c mice making progressors (tumor-bearing) and regressors (tumor-rejecting) simultaneously available. Mice bearing either an AKR kidney allograft or a sponge alone were used as controls. The cell population infiltrating the sponge was evaluated 2, 5, 10, 15, and 21 days after subcutaneous implantation. It consisted of macrophages, lymphocytes, and neutrophils. There was no difference between groups on Day 2 and Day 5. From Day 10 onwards, tumor growth was evident with a clear cut separation between progressors and regressors. The latter behaved like the two control groups except for a significant increase in lymphocytes on Day 21. The progressors showed a striking increase in total cell count from Day 10 onwards. Macrophages were the major population with a maximum value of 201 X 10(6) as compared to 8 X 10(6) in the regressors; their phagocytic and lysosomal activity remained similar in all groups. The lymphocytes showed no variation in absolute numbers but, because of the high cell count in progressors, their ratio to macrophages reached 1:50 on Day 10, making up only 2% of the cell population. Neutrophils were significantly increased in progressors as compared with the other groups. It can be concluded that in the presence of a foreign body reaction a marked influx of macrophages accompanies allogeneic tumor growth.

Murine lymphoid chalones and their in vivo effect were studied. Cytoplasmic extracts were prepared from normal murine spleen lymphocytes. When assayed in vitro, cytoplasmic extracts (CE) were able to inhibit a blastomitogenic phytohemagglutinin (PHA) response of syngeneic and allogeneic normal murine lymphocytes, as well as cell replication of murine lymphoma cells. CE also had an inhibitory effect on human lymphoblastoid cell line division but it had no effect on nonlymphoid cell division. In vivo, CE had an enhancing effect on a murine allogeneic tumor, probably by suppressing the host’s immune rejection response.

Various immunization assays were used to demonstrate the lack of immunogenicity of three BALB/c tumours of spontaneous origin and of a fourth one resulting from foreign body tumorigenesis. All four tumours inhibited the growth of a second implant of the same tumour into the contralateral flank. In our tumour models "concomitant immunity" (1) was not mediated by macrophage or T-cell dependent immune reactions: both thymectomized BALB/c and nude mice (treated or untreated with silica) gave the same results as intact mice; (2) showed some degree of non-specificity, inhibiting the growth of a different tumour in 3/4 cases; though, the existence of a specific component could not be discarded; (3) was proportional to the volume of the primary tumour at the time of the second challenge; (4) was dependent on actively growing primary tumour, not being obtained with progressively increasing daily inocula of irradiated tumour cells; (5) was detectable in an actively growing secondary tumour; recurrent growth after partial surgical excision was inhibited and (6) involved cytostasis of the secondary tumour: a syngeneic graft of the overlying skin led to tumour growth while histological studies revealed the presence of viable tumour cells. It is postulated that "concomitant immunity" or resistance can be generated without the active participation of the immune system and that tumour-related factors are, in certain cases, responsible for blocking the growth of secondary tumours. Images Figure 5

Mouse early embryos and embryo fragments were transplanted into an immunologically privileged site, consisting of a glass cylinder previously implanted under the skin of adult mice in order to test their tumor producing potential, in allogeneic adult recipients. The highest yield of tumors was obtained upon transplantation of 6 1/2 day old embryos in toto. i.e., including the embryonic and extraembryonic areas. Histological examination showed teratomas composed of differentiated tissues derived from the three germ layers containing isolated foci of undifferentiated cells and nodules of trophoblast giant cells. Areas exhibiting the histological appearance of yolk sac carcinoma were also observed. Transplantation of the whole 6 1/2 day old egg cylinder, including the ectoplacental cone, and the isolated embryonic area produced a lower incidence of teratomas with a reduced variety of differentiated tissues. No yolk sac carcinoma was found in these grafts. The ectoplacental cone of 6 1/2 day embryos produced no tumors. Grafts of genital ridges from 12 1/2 day embryos gave rise to teratomas with well differentiated tissues of embryonic and extraembryonic origin. Areas ressembling yolk sac carcinoma were also observed. The life span of trophoblastic giant cells within the glass cylinder was significantly longer than in other experimental systems.

The effects of Corynebacterinm parvum and cortisone acetate (CA) on the primary Lewis lung carcinoma and its pulmonary metastases were investigated. C. parvum given IV either on the same day or 7 days after tumour inoculation, reduced primary tumour growth, while 2.5 mg CA (high-dose) given SC 4 and 11 days after tumour, alone or in combination with C. parvum, administered on day 0, reduced primary tumour growth to the same extent as C. parvum alone. High-dose CA given on days 2 and 6 had no effect on primary tumour growth or the action of C. parvum, administered on day 7, while 0.05 mg CA (low-dose) given on days 4 and 11 did not alter tumour growth or the action of C. parvum, given at the same time as tumour. High-dose CA given 4 and 11 days after tumour caused a significant enhancement in metastases. C. parvum given to these mice on the same day as tumour significantly reduced the pulmonary nodules but only to the level found in control, saline-treated mice. In mice given C. parvum alone, metastases were significantly reduced when compared with controls. Similarly, high-dose CA given on days 2 and 6 significantly enhanced metastases, and C. parvum on day 7 reduced their level to that found in control mice. Low-dose CA had no effect on the number of metastases or the antimetastatic action of C. parvum. The relevance of these results to the clinical situation is discussed.

RNA extracted from normal liver had a specific immunodepressive effect in syngeneic BALB mice when administered 1 or 5 days before the antigen, as demonstrated in three different systems: 1) syngeneic leukemic colonization was accelerated and death anticipated; 2) spleen lymphocytes cultured in vivo within Millipore chambers showed a lower rate of spontaneous blastogenesis and lack of response to phytohemagglutinin; 3) after immunization with sheep red blood cells, spleen lymphocytes had a decreased rosette-forming capacity and the titer of hemagglutinins in serum was lower. It can be concluded that RNA pretreatment affects both cellular and humoral components of the immunologic system; the effect would be exerted on macrophages and/or lymphocytes.

Sarcoma 180 (S-180) is 100% lethal in BALB mice and leads to spontaneous regressions in AKR, A and inbred Rockland mice. Splenectomy significantly increases the regression rate of S-180 in these strains when performed either 7 days before or after the tumor challenge. That this effect is specific is suggested by the fact that partial hepatectomy has no effect and that the growth of 3 other sarcomas, either syngeneic or allogeneic, remains unaffected by splenectomy. In BALB mice, diffusion chambers with 0·22 μ filters, enclosing viable syngeneic spleen cells or S-180 cells, introduced intraperitoneally at the time of splenectomy and 7 days before S-180 challenge, are both capable of reversing the effect of splenectomy. This is not obtained in control groups including either diffusion chambers enclosing cells from a different sarcoma or empty chambers. Hypothetically these data would indicate that S-180 cells trapped within the diffusion chambers would permit the elaboration of humoral antibodies in the absence of the spleen. Upon tumor challenge, 7 days later, the presence of these antibodies would alter the temporal relationship between the humoral and cellular immune responses, favoring progressive tumor growth.

An AKR lymphoma, conditioned to grow in BALB mice by inoculating it within a subcutaneously implanted glass cylinder, led to the development of an allogeneic tumor, lymphoma P, in 65% of the animals killing them in an average of 39 days. Splenectomy performed 12 days before tumor challenge significantly decreased the incidence of lymphoma P to 40%. However, the condition of maximal tumor enhancement obtained by pretreatment of the host with soluble tumor antigen 10 days before tumor challenge remained unaltered by splenectomy, 91% of the animals dying of tumor as compared to 92% of the controls.

Concomitant resistance to a second tumor implant was induced in both conventional and nude BALB/c mice by two nonimmunogenic syngeneic tumors of spontaneous origin, an epidermoid carcinoma and a lymphoid leukemia. In the secondary tumor, which was significantly inhibited by concomitant resistance, histologic examination revealed the presence of well-preserved tumor cells without any sign of necrosis and without any host cell infiltration, contrasting with classical immunologic rejection. Tumor cell proliferation as evaluated by the number of mitoses per high-power field was significantly inhibited in the secondary tumor as compared with the corresponding controls. No effect of concomitant resistance could be detected on primary tumor growth.

In a previous paper we reported that medroxyprogesterone acetate (MPA) decreased the incidence of foreign body tumorigenesis in BALB c mice but that mammary adenocarcinomas appeared in some of the females. The experiment was repeated in 245 virgin females as follows: (1) 40 mice treated with 40 mg of MPA depot s.c. every 2 months during a whole year; (2) 117 mice bearing a foreign body (FB) and treated with MPA; (3) 46 mice bearing a FB; (4) 42 non-treated mice. Mammary adenocarcinomas developed in 16 40 in group 1 and 30 117 in group 2; no mammary tumors appeared in either control groups. The tumors were infiltrating adenocarcinomas often affecting more than one mammary gland; metastases were occasionally observed. Animals killed after 1 year of MPA treatment presented deciduomas. MPA also decreased the incidence of FB-induced sarcomas, confirming previous results.

Mammary adenocarcinomas were induced by medroxy-progesterone acetate (MPA) in female BALB/c mice. From 5 primary tumors, 9 different sublines were established by s.c. transplantation into syngeneic female mice; these developed after a long latent period (4-12 months). Each subline was transplanted both into 4 mice treated with 40mg of MPA depot (s.c. contralaterally to the tumor inoculum) and into 4 non-treated mice. Of the 9 sublines, 6 proved to be hormone-dependent (MPA-D) and 3 hormone-independent or autonomous (MPA-I). However, even the autonomous lines, when treated with MPA, showed a slight increase in growth. All MPA-D lines had a high content of ER (20-254 fmoles/mg of protein), PR (63-710), PRL-R (44-74) and low or non-detectable EGF-R. Of the 3 MPA-I sublines that were studied, 2 showed a high content of ER (16-125), PR (27-708), PRL-R (19-70) and EGF-R (29-65) while the other one had a low content of ER (0-36), PR (0-13), no EGF-R and moderate PRL-R (15-52). Spontaneous mammary tumors of BALB/c and C3H origin, which also showed an MPA-I pattern of tumor growth, had high levels of EGF-R. We postulate that MPA has a direct effect on mammary tumor cells in MPA-D lines and that the expression of EGF-R is correlated with an autonomous pattern of growth.

An attempt was made to explain the distinct lung metastatic patterns of 2 mammary adenocarcinomas with a common BALB/c origin: M3, which does not induce spontaneous metastases, and MM3 with an almost 100% incidence. No difference between the 2 tumors was detected with respect to host mononuclear cell content, degree of immunogenicity or lung-colony-forming ability. Conversely, there was a marked difference in the capacity to induce concomitant resistance: M3-bearing mice induced stronger and earlier resistance against i.v. challenge of both M3 and MM3 tumor cells than MM3-bearing mice; this resistance was expressed as lower number of lung metastases and lower tumor-cell proliferation in metastatic nodules. M3 was also able to control the development of spontaneous metastases: metastases developed in all M3-excised mice, compared with none in M3-bearing mice, while MM3-bearing mice also bearing a secondary M3 tumor developed fewer metastases than mice bearing MM3 only. This anti-metastatic effect does not appear to depend on classical immunological mechanisms since no difference could be detected between the 2 tumors in response to T cells, NK, macrophages or antibodies.

Identical and reciprocal adult F1 mice from different strain combinations, either nursed on their own mothers or foster-nursed on mothers from the paternal strain, were used to carry out SMLC assays. The results obtained showed that: (1) in vitro proliferation of F1 T cells was significantly different when splenocytes from identical versus reciprocal hybrids were used as the stimulatory population, splenocytes from one of the members of the reciprocal pair being able to induce higher proliferative responses of T cells from both identical and reciprocal F1 hybrids; (2) foster-nursing of F1 hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their splenocytes to stimulate the proliferation of responder F1 T cells. The stimulatory ability of splenocytes from foster-nursed hybrids was indistinguishable from that observed in the reciprocal F1 combination nursed by its own mother. The existence of a maternal effect acting through milk on the outcome of self recognition in the litter is discussed.

This paper reports on the investigation of the effect of medroxyprogesterone acetate (MPA) on foreign body tumorigenesis that resulted from sc implantation of a glass cylinder. Adult BALB/c mice of both sexes bearing the foreign body were separated into groups. Group 1 received 40 mg MPA sc every 2 months during 1 year, in the vicinity of the glass cylinder; group 2 received the same MPA treatment in the contralateral flank; and group 3 received no hormonal treatment. Sarcomas developed in 4 of 39, 9 of 41, and 17 of 39 mice, respectively. With the use of an evaluation based on the number of high-risk mice per time interval, the MPA inhibitory effect was found to be statistically significant in both groups: 26, 53, and 79% tumor incidence, respectively. A decrease in the rate of tumor development also was observed but only in mice treated with MPA in situ. An unexpected side effect of continuous MPA administration in females was the appearance of adenocarcinomas.