A new allele of the lpr locus, lprcg, that complements the gld gene in induction of lymphadenopathy in the mouse.

Several mice with generalized lymphadenopathy were found in the CBA/KlJms (CBA) colony maintained at our institute. A new mutant strain of mice that develop massive lymphoid hyperplasia at 100% incidence within 5 mo after birth was established by crossing these diseased mice. Genetic studies on lymphadenopathy were conducted in F1, F2, and backcross populations from crosses between mutant CBA (CBA-m) and various inbred strains of mice. The results supported the control of lymphadenopathy by a single autosomal recessive gene. Since C3H/He-gld/gld (C3H-gld), MRL/MpJ-lpr/lpr (MRL-lpr), and C3H/HeJ-lpr/lpr (C3H-lpr) mice develop the same type of lymphoid hyperplasia, allelism of the mutant gene with gld or lpr was tested by investigating lymphadenopathy in F1 and backcross populations from crosses between CBA-m and C3H-gld, MRL-lpr, or C3H-lpr mice. The gene was confirmed to be allelic with lpr but not with gld. Interestingly, however, the mutant gene interacted with gld to induce less severe lymphadenopathy. Thus, the mutant gene was named lprcg, an lpr gene complementing gld in induction of lymphoproliferation. The genetic conclusion was supported by the same profile of surface markers of lymphoid cells with gld/gld, lpr/lpr, lprcg/lprcg, lprcg/lpr, and +/gld +/lprcg genotypes, as well as by massive lymph node hyperplasia and high titers of autoantibodies in the first four genotypes, but slight hyperplasia and insignificant autoantibody production in the last. The discovery of lprcg provided strong genetic evidence for the parallels between anomalous phenotypes of gld and lpr, and CBA/KlJms-lprcg/lprcg mice will contribute to elucidation of the mechanism of induction of the same abnormal differentiation and functions of lymphocytes by gld and lpr.

Massive upregulation of the Fas ligand in lpr and gld mice: implications for Fas regulation and the graft-versus-host disease-like wasting syndrome.

Fas-deficient lpr and gld mice develop lymphadenopathy due to the accumulation of T cells with an unusual double negative (DN) (CD4-CD8-) phenotype. Previous studies have shown that these abnormal cells are capable of inducing redirected lysis of certain Fc receptor-positive target cells. Since the Fas ligand (FasL) has recently been shown to be partly responsible for T cell-mediated cytotoxicity, lymph node cells from lpr and gld mice were examined for the expression of FasL mRNA. Northern blot analysis revealed that lymph node cells obtained from lpr and gld mice had a striking increase in the level of expression of FasL mRNA predominantly due to expression in the DN T cells. Furthermore, lpr, but not gld lymph node cells killed the B cell line, A20, in a Fas-dependent manner. These findings indicate that Fas mutations result in a massive up-regulation of FasL which, most likely, results from repetitive exposure to (self) antigen. This phenomenon could explain the lpr-induced wasting syndrome observed when lpr bone marrow-derived cells are adoptively transferred to wild-type recipients.

The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer's patch high endothelial venules.

Chemokines have been hypothesized to contribute to the selectivity of lymphocyte trafficking not only as chemoattractants, but also by triggering integrin-dependent sticking (arrest) of circulating lymphocytes at venular sites of extravasation. We show that T cells roll on most Peyer's patch high endothelial venules (PP-HEVs), but preferentially arrest in segments displaying high levels of luminal secondary lymphoid tissue chemokine (SLC) (6Ckine, Exodus-2, thymus-derived chemotactic agent 4 [TCA-4]). This arrest is selectively inhibited by functional deletion (desensitization) of CC chemokine receptor 7 (CCR7), the receptor for SLC and for macrophage inflammatory protein (MIP)-3beta (EBV-induced molecule 1 ligand chemokine [ELC]), and does not occur in mutant DDD/1 mice that are deficient in these CCR7 ligands. In contrast, pertussis toxin-sensitive B cell sticking does not require SLC or MIP-3beta signaling, and occurs efficiently in SLC(low/-) HEV segments in wild-type mice, and in the SLC-negative HEVs of DDD/1 mice. Remarkably, sites of T and B cell firm adhesion are segregated in PPs, with HEVs supporting B cell accumulation concentrated in or near follicles, the target domain of most B cells entering PPs, whereas T cells preferentially accumulate in interfollicular HEVs. Our findings reveal a fundamental difference in signaling requirements for PP-HEV recognition by T and B cells, and describe an unexpected level of specialization of HEVs that may allow differential, segmental control of lymphocyte subset recruitment into functionally distinct lymphoid microenvironments in vivo.

Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.

Secondary lymphoid organ chemokine (SLC) is expressed in high endothelial venules and in T cell zones of spleen and lymph nodes (LNs) and strongly attracts naive T cells. In mice homozygous for the paucity of lymph node T cell (plt) mutation, naive T cells fail to home to LNs or the lymphoid regions of spleen. Here we demonstrate that expression of SLC is undetectable in plt mice. In addition to the defect in T cell homing, we demonstrate that dendritic cells (DCs) fail to accumulate in spleen and LN T cell zones of plt mice. DC migration to LNs after contact sensitization is also substantially reduced. The physiologic significance of these abnormalities in plt mice is indicated by a markedly increased sensitivity to infection with murine hepatitis virus. The plt mutation maps to the SLC locus; however, the sequence of SLC introns and exons in plt mice is normal. These findings suggest that the abnormalities in plt mice are due to a genetic defect in the expression of SLC and that SLC mediates the entry of naive T cells and antigen-stimulated DCs into the T cell zones of secondary lymphoid organs.

Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci.

In MRL mice, the mostly recessive lpr mutation results in both the accumulation of CD4-, CD8-, CD3+ T cells in lymphoid tissue and many features of generalized autoimmune disease, including immune complex glomerulonephritis. To positionally clone the lpr mutation and analyze the effects of background genes, backcross offspring were examined from the cross: (MRL/MpJ-lpr x CAST/Ei)F1 x MRL/MpJ-lpr. The lpr gene was found to be closely linked to a mouse chromosome 19 marker defined by a variation of a Fas gene restriction fragment. Our results identified differences in RNA expression and differences in the genomic organization of the Fas gene between normal and lpr mice, and confirm the recent report that a mutation in the Fas apoptosis gene is the lpr mutation. However, our results also indicate that the Fas gene is expressed in spleen cells from normal mice, and spleen and lymph node cells from mice with a second mutation at the lpr locus (lprcg). Together these results suggest that altered Fas transcription results in the failure of lymphocytes to undergo programmed cell death and may lead to an altered immune cell repertoire. This mechanism may explain certain central and peripheral defects in tolerance that are present in autoimmune disease. The current study also demonstrates the profound effect of background genes on the degree of nephritis, lymphadenopathy, and anti-DNA antibody production. Of major note, our studies suggest the identification of chromosomal positions for genes that modify nephritis. Analysis of the backcross mice for markers covering most of the mouse genome suggests that over 50% of the variance in renal disease is attributable to quantitative trait loci on mouse chromosomes 7 and 12. Moreover, this study provides a model for dissecting the complex genetic interactions that result in manifestations of autoimmune disease.

The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules.

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function-associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Galpha(i)-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (T(GFP) cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling T(GFP) cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked T(GFP) cell adherence in wild-type HEVs, whereas desensitization to stromal cell-derived factor (SDF)-1alpha (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect T(GFP) cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, T(GFP) cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1-mediated T(GFP) cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Lack of growth of a pregnancy-dependent mouse mammary tumor (TPDMT-4) in the absence of pituitary hormones.

Mammary tumors of line TPDMT-4, established in DDD mice, were characterized by growth during pregnancy and regression after parturition; this resulted in higher growth peaks in subsequent pregnancies in breeders and no growth in virgins. The effect of hypophysectomy on tumor growth in mice given 17beta-estradiol (E) and progesterone (P) or deoxycorticosterone acetate (DCA) was investigated. Growth of cancers occurred in E+P- and E+DCA-treated virgins, but not in cholesterol-treated virgins. Tumors did not grow to palpable sizes in cholesterol-, E+P-, and E+DCA-treated hypophysectomized virgins; this indicated that pituitary hormones were essential for tumor growth. Impalpable cholesterol-treated, 5 of 10 E+P-treated, and 3 of 6 E+DCA-treated hypophysectomized animals. The neoplasms showed ductal and tubular structures that were lined by a single layer of well-differentiated buoidal epithelium, which suggested that the tumor line might be derived from ductal cells.

Response of a pregnancy-dependent mouse mammary tumor to hormones.

A transplantable, pregnancy-dependent, mouse mammary tumor line (TPDMT-4) showed a pregnancy-dependent growth in DDD breeders. The tumors grew during pregnancy, regressed rapidly after parturition, and reached ascending peaks in subsequent pregnancies. Growth without regression occurred in animals with pituitary isografts (PI), but not after the hosts were ovariectomized. The effects of ovariectomy was negated by sc injections of both 17beta-estradiol (E) and progesterone (P), but single injections of E or P did not abolish the effect of the ovariectomy. The tumors also grew in virgins given sc implants of E and P or deoxycorticosterone acetate (DCA) pellets. Adrenalectomy had no influence on the tumor growth in PI-bearing animals, but DCA injections stimulated secretion production by both tumor cells and normal mammary cells. The hormonal conditions that produced tumor growth caused in the host mammary glands the full lobulo-alveolar development seen in mid- to late-pregnant animals. In the hosts without tumor growth, the mammary glands had small clusters of acini at most. The tumors were classified as type A morphologically. Our results suggest that the growth of TPDMT-4, like that of normal mammary glands, is controlled by prolactin, E, and P.

Trisomy of chromosome No 13 in spontaneous mammary tumors of GR, C3H, and noninbred Swiss mice.

Karyotype analyses were conducted on spontaneous mammary tumors of 11 GR, 2 C3H, and 2 noninbred Swiss mice with the use of trypsin-Giemsa banding procedures. All tumors manifested trisomy of chromosome No 13 in most cells, and all except 1 tumor had cells with a model chromosome number of 40.

Genetic resistance in Japanese wild mice (Mus musculus molossinus) to an NB-tropic Friend murine leukemia virus.

Wild mice (Sk, Hz-Vl, Hz-IV Om, Mol.A, Fu, Te, and Sn) trapped in various areas of Japan were crossed with mice of inbred strains (C57BL/6, C57L, BALB/c, and C57BL/10), and their progeny were infected with NB-tropic Friend nurine leukemia virus. Ten days after infection, the spleens were weighed, examined for macroscopic focal lesions, and assayed for infectious virus by the XC test. Genetic analysis indicated that 4 of 8 mice tested had a dominant gene that suppresses the virus replication; the gene resembles the Fv-4' allele. No mice with the Fv-2' allele were found.

Differential response of an ovarian-responsive mouse mammary tumor to androgenic and estrogenic agents.

An ovarian-responsive mammary tumor subline, T4-OR26, was isolated from an outgrowth of a progressed TPDMT-4 pregnancy-dependent mammary tumor in a virgin DDD mouse. T4-OR26 tumors were characterized by significantly faster growth in virgin mice than in ovariectomized mice. Both estrogen and progesterone were important for growth of the subline, as they were for that of the parent. Tamoxifen (TAM), with estrogenic activity, and epithiostanol (EPI) and testosterone propionate, with androgenic activity, which all caused TPDMT-4 tumors to regress, were compared for antitumor potency against the new subline by 3 s.c. injections weekly in virgins. EPI at 300 micrograms and testosterone propionate at 1000 micrograms elicited immediate tumor growth suppression with subsequent slight regression as did ovariectomy. TAM at 1000 micrograms caused tumor growth suppression after 2 weeks without subsequent regression. At 600 micrograms, EPI but not TAM significantly inhibited 17 beta-estradiol plus progesterone-induced tumor growth; at 400 micrograms, neither had any significant effect on the tumor growth induced by 17 beta-estradiol alone. With regard to their effect on hormone receptors, it was noted that EPI and testosterone propionate treatments with tumor regression caused significant reduction in cytoplasmic progesterone receptor, but TAM treatment, which does not influence tumor growth, did not cause such reduction. The results provide evidence that hormone-dependent mammary tumors may acquire greater resistance to estrogenic than to androgenic therapeutics with progression.

Inhibited growth in vivo of a mouse pregnancy-dependent mammary tumor (TPDMT-4) by an antiestrogen, 2alpha, 3alpha-epithio-5alpha-androstan-17beta-ol (10275-S).

TPDMT-4 mammary tumors, characterized by growth during pregnancy and postpartum regression, grew continuously in DDD virgin mice carrying pituitary isografts or 17 beta-estradiol plus progesterone pellets. In the experimental models, effects of 2alpha,3alpha-epithio-5alpha-androstan-17beta-ol(10275-S), testosterone, and ovariectomy were investigated. When tumors implanted with pituitary isografts into the fat pad reached palpable volumes, animals were ovariectomized or received 5 s.c. injections of 100 mug to 1 mg 10275-S or 300 mug testosterone weekly. Tumors regressed immediately after ovariectomy or after the start of 10275-S treatment and after approximately the 10th day of testosterone treatment. Cystadenomatous morphology with strong secretory activity was noticed in treated animals. Tumor growth induced by 17beta-estradiol plus progesterone pellets was inhibited completely or partially by 3 s.c. injections of 50 to 500 mug 10275-S or 100 mug testosterone weekly starting from the day after tumor and 17beta-estradiol plus progesterone pellet implantation.

Antitumor effect of two oral steroids, mepitiostane and fluoxymesterone, on a pregnancy-dependent mouse mammary tumor (TPDMT-4).

Pregnancy-dependent TPDMT-4 mammary tumors, characterized by requiring estrogen, progesterone, and pituitary hormones for growth, grew continuously in female DDD mice carrying pituitary isografts. The experimental model was used to investigate the antitumor effects of two p.o. steroids, mepitiostane and fluoxymesterone. When tumors implanted with pituitary isografts into the fat-pad reached palpable size, animals received 6 doses/week of 0.1, 0.3, 1.0, and 3.0 mg of either steroid intragastrically. Mepitiostane significantly suppressed tumor growth with regression in 25 and 29 percent of animals at 1.0 and 3.0 mg, respectively, but had no inhibitory effects at other doses. Fluoxymesterone retarted tumor growth during the first week of treatment at 3.0 mg but finally had no inhibitory effects at any doses. Under similar conditions ovariectomy caused tumor regression immediately, and epitiostanol, the parent steroid of mepitiostane, significantly suppressed tumor growth when given in 3 injections/week of 0.5 mg s.c. Tumous had papillary structures and almost lacked secretory activity. A comparison of these findings to those obtained with earlier generations suggests that TPDMT-4 tumors became less sensitive to the antitumor effect of epitiostanol and were able to grow at lower hormone levels with succeeding generations. Morphologically, progression to more cancer and less secretory activity was noticed.

Accelerated progression to autonomy of a pregnancy-dependent mouse mammary tumor (TPDMT-4) by hormones.

TPDMT-4 mouse mammary tumors have been characterized by pregnancy-dependent growth in breeders; formation of ductal, lobular, and alveolar structures in gland-free but insignificant outgrowth in intact fat pads of virgins; and continuous growth in hosts implanted s.c. with estrogen-progesterone pellets. Tumors were passaged in breeding and estrogen-progesterone-treated mice for 30 and 28 generations, respectively. The latter subline was called TPDMT-4EP. Both lines were compared for tumor-producing capability in virgins. Transplants from growing TPDMT-4 and growing and regressing TPDMT-4EP tumors were implanted into cleared and intact fat pads of 3-week-old mice and investigated for tumor formation for 12, 4, and 4 months, respectively. No significant differences were noted between cleared and intact fat pads. TPDMT-4 transplants formed significant tumors in 26 (72%) of 36 fat pads after a mean latency period of of 204 days. Growing and regressing TPDMT-4EP transplants formed significant tumors in 47 (98%) of 48 and in 27 (90%) of 30 fat pads, respectively, after a mean latency period of 52 days. Of eight TPDMT-4 tumors tested for ovarian dependence, six were ovarian dependent, one was ovarian responsive, and one was undecided. In contrast, of 31 TPDMT-4EP tumors tested, three were ovarian responsive, and 28 were ovarian independent. Five of seven independent tumors from ovariectomized mice still responded to the estrogen-progesterone pellet. Cytoplasmic estrogen and progesterone receptors were assayed in tumors different in ovarian dependence without noting significant correlation between their levels and ovarian dependence or growth rate. The results suggest that continuous hormone stimulation may enhance progression from dependence to autonomy of hormone-dependent tumors.

Antitumor effect of the antiestrogen, tamoxifen, ona pregnancy-dependent mouse mammary tumor (TPDMT-4).

TPDMT-4 mammary tumors, characterized by growth during pregnancy and regression after delivery, show continued growth in female DDD mice carrying pituitary isografts ectopically or a s.c. 17 beta-estradiol-plus-progesterone pellet. These experimental models were used to investigate the antitumor effect of the nonsteroidal antiestrogen tamoxifen. When tumors grew to significant sizes, tamoxifen was injected s.c. at a daily dose of 800, 400, 200, 100, 50, 5 or 1 micrograms three times weekly. In pituitary isograft-bearing mice the antitumor effect of tamoxifen at the three highest doses was comparable to that of ovariectomy: tumors regressed slowly during the first 2 weeks of treatment and subsequently more rapidly. Tamoxifen at 100 and 50 micrograms had no influence on tumor growth during the first 2 weeks and subsequently gave rise to rapid regression. The antiestrogen suppressed tumor growth throughout the treatment period at 10 micrograms, but it had no effect on growth at the two lowest doses. Tamoxifen caused atrophy of the ovaries and mammary glands in a dose-related manner, that of luteal components in the former being especially conspicuous at 200-micrograms doses and higher. In pellet-carrying mice, tamoxifen suppressed tumor growth completely at 800 micrograms but partially and insignificantly at 200 micrograms. Growth of PIMT-16 autonomous mammary tumors included for comparison was not affected either by tamoxifen at 800 micrograms daily or by ovariectomy. Tamoxifen is suggested to exert an antitumor effect on this particular hormone-dependent mammary tumor model through its direct action on tumor cells and its suppressive action on hormone production by the ovaries and pituitary gland.

Augmentation of antitumor immunity with bacterial superantigen, staphylococcal enterotoxin B-bound tumor cells.

We have examined the effectiveness of a bacterial superantigen, staphylococcal enterotoxin B (SEB)-coupled tumor cells, to induce antitumor activity. SEB was chemically conjugated to tumor cells using a heterobifunctional cross-linking agent acting through NH2 and SH groups. V beta 8+ T cells were activated and increased in number after the culture with SEB-bound Meth A cells. The cultured T cells exhibited an antitumor activity in the Winn assay. Interleukin 2 (IL-2) receptor alpha + (IL-2R+) V beta 8+ T cells but not IL-2R+ V beta 6+ T cells increased in number in mice injected with SEB-bound Meth A cells. However, the percentages of V beta 8+ and V beta 6+ T cells did not change by this immunization. The antitumor effector cells were V beta 7- 8- CD4+ T cells. In vivo immunization with SEB-bound cells induced a strong antitumor activity, i.e., tumor-free mice/total mice = 14 of 15 (93%) for Meth A and 7 of 15 (47%) for hepatoma MH134. The induced antitumor activity was both dose dependent and tumor specific. Treatment with SEB-bound cells prolonged the survival days of Meth A-bearing mice by 62%. These results suggest that SEB-bound tumor cells may be a powerful method for induction of in vivo antitumor activity.

Pregnancy-dependent to ovarian-independent progression in mammary tumors delineated in primary culture: changes in signal transduction, growth factor regulation, and matrix interaction.

A model for the evolution of hormone-independent tumors from a pregnancy-dependent precursor is exemplified by the TPDMT-4[pregnancy-dependent (PD)] and T4OI96 [ovarian-independent (OI)] in vivo tumor lines developed in DDD mice. In vivo, the OI tumor grows rapidly in virgin mice and is more anaplastic than the PD tumor which, in virgin mice, grows as a hyperplastic alveolar gland from which tumors arise only during pregnancy. The regulation of the proliferation of these two tumors was compared in primary culture using a three-dimensional, serum-free, collagen gel cell culture system. In medium containing insulin, the growth of cell organoids from PD tumors was stimulated by the same factors that stimulate the growth of normal mammary epithelial cells from virgin or pregnant mice. These factors include progesterone and prolactin (but not estrogen), epidermal growth factor, basic fibroblast growth factor, linoleic acid, cyclic AMP, prostaglandin E2, phosphatidic acid, and lithium. Most of these tumors (about 80%) could not grow in medium with only insulin present; of those that did, growth was slow and was stimulated further by the above agents. PD tumor cells formed stellate colonies in the collagen matrix similar to those of normal cells. These findings show that the growth regulation of PD tumor cells is, in all aspects examined, similar to that of normal cells. In addition, the capacity for growth in the presence of only insulin (more autonomous growth) is not necessarily accompanied by deletions in responses to growth factors or hormones, or in lipid response pathways. In contrast, organoids from OI tumors needed only insulin for growth. The growth of some of these tumors was stimulated further by only basic fibroblast growth factor and phosphatidic acid. Cyclic AMP and agents that elevate intracellular cyclic AMP inhibited growth, the opposite of the response seen in normal or PD cells. OI organoids grew as cell masses rather than as stellate structures. These data show that while PD tumors are remarkably similar to normal cells in the regulation of their proliferation, OI tumors have incurred multiple defects in growth-regulatory pathways possibly including the production of autocrine growth factor(s). In addition, the ability of OI tumor cells to adhere to the collagen gel matrix and undergo normal morphogenesis is reduced. These results may highlight specific events required for the progression from ovarian dependency to ovarian independency related to the hormonal regulation of growth.

Cytoplasmic estrogen receptor in a pregnancy-dependent mouse mammary tumor (TPDMT-4) and related autonomous lines.

Transplantable TPDMT-4 mammary tumors are characterized by pregnancy dependence, growth during pregnancy, regression after delivery, and practically no growth in virgin animals. In this study, tumor pieces were treated with chemical carcinogens in vitro and serially transplanted in virgin mice with increasingly short intervals between passages. Two autonomous sublines were obtained utilizing 4-nitroquinoline 1-oxide (1 microgram/ml) and 20-methylcholanthrene (10 microgram/ml) and were designated as PIMT-4491 and PIMT-4673, respectively. PIMT-4673 tumors, very similar morphologically to the parent tumors, were composed of small cuboidal epithelial cells which occasionally formed acinar and glandular structures. PIMT-4491 showed similar morphology with groups of spindle cells at early generations but became more sarcomatous at later generations. Cytoplasmic estrogen receptor (ER) was examined by sucrose density and dextran-coated charcoal methods in newly established and parent tumors. ER was undetectable in PIMT-4673. PIMT-4491 had the same levels of ER as did parent tumors up to generation 21, when ER levels began to decrease gradually until they reached 5.1 fmol/mg cytosol protein at generation 50. In TPDMT-4, ER levels varied from 20 to 75 fmol/mg cytosol protein and were not related to growth behaviors. Effects on TPDMT-4 tumors of estradiol and progesterone alone or in combination were investigated at generations 1- and 33. Under all hormonal conditions, late-generation tumors grew better than early ones. ER levels were not significantly different in the two generations, although they tended to be slightly higher at the later generation. Apparent dissociation constants estimated by Scatchard plots were on the order of 10(-10) M in all ER-positive tumors.

Down regulation of Bcl-2 is the first step on Fas-mediated apoptosis of male reproductive tract.

Suppression of apoptosis by Bcl-2, an oncogene product, has been previously reported. Although the down regulation of Bcl-2 has been encountered in various types of apoptosis, the time course of changes in the expression of Bcl-2 has yet to be determined. In the present study, we established and analysed an in vivo model of apoptosis. The mouse male reproductive tract, specifically the prostate and epididymis, which is regulated by sex steroid hormones, especially testosterone, showed regression induced by chromosomal DNA fragmentation, apoptotic cell death, after gonadectomy. Following this apoptosis, down regulation of Bcl-2 and the new expression of Fas were seen. Using functional Fas-lacking mice, we demonstrated that this regression of the male reproductive tract is triggered by Fas-mediated apoptosis. In addition, time course experiments revealed that down regulation of Bcl-2 when apoptosis occurs heralds Fas expression. We propose here that apoptotic death signal transduction in total involve, a number of steps, the first and most important of which is down regulation of Bcl-2.