Galectin-1, -3 and -9 Expression and Clinical Significance in Squamous Cervical Cancer

Galectins are proteins that bind β-galactoside sugars and provide a new type of potential biomarkers and therapeutic targets in cancer. Galectin-1, -3 and -9 have become the focus of different research groups, but their expression and function in cervical cancer is still unclear. The aim of this study was to determine the phenotype of galectin-1, -3 and -9 expressing cells and the association with clinico-pathological parameters in cervical cancer. Galectin expression was scored in tumor cells, tumor epithelium infiltrating immune cells and stromal cells in squamous cervical cancer (n = 160). Correlations with clinico-pathological parameters and survival were studied according to the REMARK recommendations. We additionally investigated whether the galectins were expressed by tumor cells, fibroblasts, macrophages and T cells. Galectin-1 and -9 were both expressed by tumor cells in 11% of samples, while 84% expressed galectin-3. Strong galectin-1 expression by tumor cells was an independent predictor for poor survival (hazard ratio: 8.02, p = 0.001) and correlated with increased tumor invasion (p = 0.032) and receiving post-operative radiotherapy (p = 0.020). Weak and positive tumor cell galectin-3 expression were correlated with increased and decreased tumor invasion, respectively (p = 0.012). Tumor cell expression of galectin-9 showed a trend toward improved survival (p = 0.087). The predominant immune cell type expressing galectin-1, -3 and -9 were CD163⁺ macrophages. Galectin-1 and -3 were expressed by a minor population of T cells. Galectin-1 was mainly expressed by fibroblasts in the tumor stroma. To conclude, while tumor cell expression of galectin-9 seemed to represent a beneficial response, galectin-1 expression might be used as a marker for a more aggressive anti-cancer treatment.     

Galectin-7 in the control of epidermal homeostasis after injury

Galectins, a family of β-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7–deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7–deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7–deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.     

Galectin-3, a Novel Centrosome-associated Protein,Required for Epithelial Morphogenesis

Galectin-3 is a β-galactoside–binding protein widely expressed in all epithelia where it is involved in tissue homeostasis and cancer progression. We recently reported unique abnormalities in the identity of membrane domains in galectin-3 null mutant mice, suggesting that galectin-3 may participate in epithelial polarity program. We investigated the potential role of galectin-3 on early events in polarization of epithelial renal cells, using three-dimensional cultures of MDCK cells and also galectin-3 null mutant mouse kidneys. We show that depletion in galectin-3 systematically leads to severe perturbations of microtubular network associated with defects in membrane compartimentation, both in vitro and in vivo. Moreover, the absence of galectin-3 impinges on the morphology of the primary cilium, which is three times longer and unusually shaped. By immunological and biochemical approaches, we could demonstrate that endogenous galectin-3 is normally associated with basal bodies and centrosomes, where it closely interacts with core proteins, such as centrin-2. However, this association transiently occurs during the process of epithelial polarization. Interestingly, galectin-3–depleted cells contain numerous centrosome-like structures, demonstrating an unexpected function of this protein in the formation and/or stability of the centrosomes. Collectively, these data establish galectin-3 as a key determinant in epithelial morphogenesis via its effect on centrosome biology.     

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.     

Binding of Toxoplasma gondii Glycosylphosphatidylinositols to Galectin-3 Is Required for Their Recognition by Macrophages

We showed that the production of tumor necrosis factor (TNF) α by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-like receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a β-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs shows that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of TNF-α induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages.     

Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a p53-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-κB activity and was inhibited by NF-κB inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-κB activity. The implication of NF-κB was further supported by data showing that NF-κB bound to the endogenous galectin-7 promoter and that TNFα-induced galectin-7 expression was abolished by NF-κB inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.     

X-ray Structures of Human Galectin-9 C-terminal Domain in Complexes with a Biantennary Oligosaccharide and Sialyllactose

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16–0.70 μm) and linear β1–3-linked poly-N-acetyllactosamines (0.09–8.3 μm) and significant affinity for the α2–3-sialylated oligosaccharides (17–34 μm). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2–3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2–3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg²²¹ is responsible for recognizing sialylated oligosaccharides.     

Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.     

Galectin-3 modulates phagocytosis-induced stellate cell activation and liver fibrosis in vivo

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)β(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)β(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.     

Galectin-3 as a Marker and Potential Therapeutic Target in Breast Cancer

Galectin-3 has a relatively high level of expression in triple-negative breast cancers and is a potential marker for this disease. However, the clinical and prognostic implications of galectin-3 expression in breast cancer remain unclear. We examined mastectomy specimens from 1086 breast cancer cases and matching, adjacent non-cancerous tissues using immunohistochemistry. Overall, triple-negative breast cancers expressed galectin-3 more strongly than did other breast cancers types (63.59% vs 21.36%, P = 0.001). Galectin-3 expression was not found to be an independent prognostic factor for breast cancer by Cox regression analysis, but was associated with chemotherapeutic resistance. Apoptosis was only weakly induced by arsenic trioxide (ATO) treatment in galectin-3-positive breast cancer cells (MDA-MB-231 and MCF-7), although ATO treatment up-regulated galectin-3 expression. Knockdown of galectin-3 in MDA-MB-231 cells sensitized them to killing by ATO. These findings support a possible role for galectin-3 as a marker for triple-negative breast cancer progression and as a therapeutic target in combination with ATO treatment, although the mechanisms that underlie this synergy require further investigation.     

Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.     

Corpus luteum susceptibility to prostaglandin F2α (PGF2α) luteolysis in hysterectomized prepuberal and mature gilts

The susceptibility of induced corpora lutea (CL) of prepuberal gilts and spontaneously formed CL of mature gilts to prostaglandin F_2α (PGF_2α) luteolysis was studied. Prepuberal gilts (120 to 130 days of age) were induced to ovulate with Pregnant Mare Serum Gonadotropin and Human Chorionic Gonadotropin (HCG). The day following HCG was designated as Day 0. Mature gilts which had displayed two or more estrous cycles of 18 to 22 days were used (onset of estrus = Day 0). Gilts were laparotomized on Day 6 to 9, their CL marked with sterile charcoal and totally hysterectomized. On Day 20, gilts were injected IM with either distilled water (DW), 2.5 mg PGF_2α or 5.0 mg PGF_2α. An additional group of prepuberal gilts was injected with 1.25 mg PGF_2α, a dose of PGF_2α equivalent, on a per kilogram body weight basis, to the 2.5 mg PGF_2α dose given to the mature gilts. The percentages of luteal regression on Day 27 to 30 for mature and prepuberal gilts given DW, 2.5 mg PGF_2α and 5.0 mg PGF_2α were 0.0 vs 4.4, 43.5 vs 96.8 and 47.7 vs 91.6, respectively; the percentage of luteal regression for the prepuberal gilts given 1.25 mg PGF_2α was 75.1. These results indicate that induced CL of the prepuberal gilt were more susceptible to PGF_2α luteolysis than spontaneously formed CL of the mature gilt and that pregnancy failure in the prepuberal gilt could be due to increased susceptibility of induced CL to the natural luteolysin.     

Galectin-3 and metastasis

Galectin-3, a 31 kDa member of the β-galactoside-binding proteins, is an intracellular and extracellular lectin which interacts with intracellular glycoproteins, cell surface molecules and extracellular matrix proteins. Galectin-3 is expressed widely in epithelial and immune cells and its expression is correlated with cancer aggressiveness and metastasis. Galectin-3 is involved in various biological phenomena including cell growth, adhesion, differentiation, angiogenesis and apoptosis. Recent research revealed that galectin-3 is associated with several steps of invasion and metastasis, like angiogenesis, cell-matrix interaction, dissemination through blood flow and extravasation. Recently, we and others have shown that galectin-3 can be a reliable diagnostic marker in certain cancers and one of the target proteins of cancer treatment. In this review, we describe the involvement of galectin-3 in each steps of metastasis and clinical significance of galectin-3. Published in 2004.     

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.     

Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch

In nonpregnant and pregnant dogs the corpora lutea (CL) are the only source of progesterone (P4) which shows an almost identical secretion pattern until the rapid decrease of P4 prior to parturition. For the nonpregnant dog clear evidence has been obtained that physiological luteal regression is devoid of a functional role of the PGF2α-system and seems to depend on the provision of StAR. Yet in pregnant dogs the rapid prepartal luteal regression, coinciding with an increase of PGF2α, may be indicative for different regulatory mechanisms. To assess this situation and by applying semi-quantitative Real Time (Taq Man) RT-PCR, expression patterns were determined for the following factors in CL of pregnant and prepartal dogs and of mid-pregnant dogs treated with the antiprogestin Aglepristone: cyclooxygenase 2 (Cox2), prostaglandin E2 synthase (PGES), prostaglandin F2α synthase (PGFS), its receptors (EP2, EP4 an FP), the steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid-dehydrogenase (3βHSD) and the progesterone receptor (PR). Peripheral plasma P4 concentrations were determined by RIA. CL were collected via ovariohysterectomy from pregnant bitches (n = 3–5) on days 8–12 (Group 1, pre-implantation period), days 18–25 (Group 2, post-implantation period), days 35–40 (Group 3, mid-gestation period) and during the prepartal progesterone decline (Group 4). Additionally, CL were obtained from groups of 5 mid-pregnant dogs (days 40–45) 24 h, respectively 72 h after the second treatment with Aglepristone. Expression of Cox2 and PGES was highest during the pre-implantation period, that of PGFS and FP during the post-implantation period. EP4 and EP2 revealed a constant expression pattern throughout pregnancy with a prepartal upregulation of EP2. 3βHSD and StAR decreased significantly from the pre-implatation period to prepartal luteolysis, it was matched by the course of P4 concentrations. Expression of the PR was higher during mid-gestation and prepartal luteolysis than in the two preceding periods. After application of Aglepristone the overall mRNA-expression resembled the situation during prepartal luteolysis except for EP2, which remained unchanged.These data suggest that – as in the nonpregnant bitch – also in the pregnant bitch luteal production of prostaglandins is associated with luteal support rather than luteolysis. On the other hand induction of luteolysis by the PR blocker Aglepristone points to a role of luteal P4 as an autocrine factor in a positive loop feedback system controlling the availability of P4, StAR and 3βHSD.     

Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.     

Treatment of multidrug resistant (MDR1) murine leukemia with P-glycoprotein substrates accelerates the course of the disease

The prolactin (PRL) surge in cycling rats during the proestrous afternoon is an inducer of apoptotic cell death in luteal cells. This luteolytic action of PRL is peculiar, because PRL may be categorized as a survival factor, if other known physiological functions of PRL are taken into account. Here we analyzed the underlying molecular/cellular mechanisms of this PRL-induced apoptosis. Corpora lutea (CL) were prepared from the ovary on the proestrous day and cultured with or without PRL (2 microg/ml). An addition of PRL to the culture medium induced DNA breakdown in the nuclei of cells mostly identified as steroidogenic by 3beta-HSD activity staining, and the number of 3beta-HSD-positive cells were significantly decreased, indicating the induction of apoptotic cell death by PRL among luteal cells in culture. Next, the expression of membrane form-Fas ligand (mFasL) in the luteal cell lysate was quantified, because Fas receptor is known to have an exact physiological role in luteolysis. An addition of PRL increased the expression of mFasL. Immunostaining and TUNEL assay on regressing CL revealed that both CD3-positive cells and FasL-positive cells were co-localized in the regions where apoptosis convergently occurred. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, to the culture mimicked the PRL action by inducing apoptosis in luteal cells and enhancing the expression of mFasL. These data suggest that the CD3-positive T lymphocyte in the CL is at least one of the PRL-effector cell species during the process of luteolysis in rats, and that FasL expression of these cells is upregulated by PRL.