Preferential expression of cystein-rich secretory protein-3 (CRISP-3) in chronic pancreatitis

BACKGROUND: Chronic pancreatitis (CP) is a progressive inflammatory process resulting in exocrine and endocrine pancreatic insufficiency in advanced stages. Cysteine-rich secretory protein (CRISP-3) has been identified as a defense-associated molecule with predominant expression in the salivary gland, pancreas and prostate. AIMS: In this study, we investigated CRISP-3 expression in normal pancreatic tissues, chronic pancreatitis tissues, pancreatic cancer tissues and pancreatic cancer cell lines, as well as in other gastrointestinal organs. MATERIALS AND METHODS: 15 normal pancreatic tissues, 14 chronic pancreatitis tissues and 14 pancreatic cancer tissues as well as three pancreatic cancer cell lines were analyzed. Moreover, hepatocellular carcinoma and esophageal, stomach and colon cancers were also analyzed together with the corresponding normal controls. RESULTS: CRISP-3 was expressed at moderate to high levels in chronic pancreatitis tissues and at moderate levels in pancreatic cancer tissues but at low levels in normal pancreatic tissues, and was absent in three pancreatic cancer cell lines. CRISP-3 expression was below the level of detection in all cancerous gastrointestinal tissues and in all normal tissues except 2 of 16 colon tissue samples. CRISP-3 mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells and in small proliferating ductal cells in CP tissues and CP-like lesions in pancreatic cancer tissues. In contrast, CRISP-3 expression was weak to absent in the cytoplasm of cancer cells as well as in acinar cells and ductal cells in pancreatic cancer tissues and normal pancreatic tissues. CONCLUSION: These results reveal that the distribution of CRISP-3 in gastrointestinal tissues is predominantly in the pancreas. High levels of CRISP-3 in acinar cells dedifferentiating into small proliferating ductal cells in CP and CP-like lesions in pancreatic cancer suggests a role of this molecule in the pathophysiology of CP.     

Gene cloning of immunogenic antigens overexpressed in pancreatic cancer

The serological analysis of recombinant cDNA expression libraries (SEREX) by utilizing a library derived from a human pancreatic adenocarcinoma cell line and IgG antibodies from an allogeneic patient serum led to the identification of 18 genes: 13 of these were known genes, and 5 were unknown genes. In Northern and RT-PCR analyses, we found that the expression of mRNA of 14 genes was elevated in pancreatic cancer cell lines compared with the levels in normal pancreatic tissues. In addition, the expression of mRNA of hsp105 in colon cancer was greater than that in normal colon tissue. Immunohistochemical analysis using anti-hsp105 antibody revealed that an increased expression of hsp105 is a characteristic feature of pancreatic ductal and colon adenocarcinoma. Furthermore, hsp105 immunoreactivity in some cases of gastric, esophageal, and hepatocellular carcinoma was much stronger than that in normal corresponding tissues. These molecules identified may provide good diagnostic markers for cancer cells.     

Epiregulin is Up-regulated in pancreatic cancer and stimulates pancreatic cancer cell growth

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.     

The effect of hypophysectomy on pancreatic islet hormone and insulin-like growth factor I content and mRNA expression in rat

The growth arrest after hypophysectomy in rats is mainly due to growth hormone (GH) deficiency because replacement of GH or insulin-like growth factor (IGF) I, the mediator of GH action, leads to resumption of growth despite the lack of other pituitary hormones. Hypophysectomized (hypox) rats have, therefore, often been used to study metabolic consequences of GH deficiency and its effects on tissues concerned with growth. The present study was undertaken to assess the effects of hypophysectomy on the serum and pancreatic levels of the three major islet hormones insulin, glucagon, and somatostatin, as well as on IGF-I. Immunohistochemistry (IHC), in situ hybridization (ISH), radioimmunoassays (RIA), and Northern blot analysis were used to localize and quantify the hormones in the pancreas at the peptide and mRNA levels. IHC showed slightly decreased insulin levels in the cells of hypox compared with normal, age-matched rats whereas glucagon in cells and somatostatin in cells showed increase. IGF-I, which localized to cells, showed decrease. ISH detected a slightly higher expression of insulin mRNA and markedly stronger signals for glucagon and somatostatin mRNA in the islets of hypox rats. Serum glucose concentrations did not differ between the two groups although serum insulin and C-peptide were lower and serum glucagon was higher in the hypox animals. These changes were accompanied by a more than tenfold drop in serum IGF-I. The pancreatic insulin content per gram of tissue was not significantly different in hypox and normal rats. Pancreatic glucagon and somatostatin per gram of tissue were higher in the hypox animals. The pancreatic IGF-I content of hypox rats was significantly reduced. Northern blot analysis gave a 2.6-, 4.5-, and 2.2-fold increase in pancreatic insulin, glucagon, and somatostatin mRNA levels, respectively, in hypox rats, and a 2.3-fold decrease in IGF-I mRNA levels. Our results show that the fall of serum IGF-I after hypophysectomy is accompanied by a decrease in pancreatic IGF-I peptide and mRNA but by partly discordant changes in the serum concentrations of insulin and glucagon and the islet peptide and/or mRNA content of the three major islet hormones. It appears that GH deficiency resulting in a low IGF-I state affects translational efficiency of these hormones as well as their secretory responses. The maintenance of normoglycemia in the presence of reduced insulin and elevated glucagon serum levels, both of which would be expected to raise blood glucose, may result mainly from the enhanced insulin sensitivity, possibly due to GH deficiency and the subsequent decrease in IGF-I production.     

Doublecortin-Like Kinase 1 Is Elevated Serologically in Pancreatic Ductal Adenocarcinoma and Widely Expressed on Circulating Tumor Cells

Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.     

Expression analysis of PMP22/Gas3 in premalignant and malignant pancreatic lesions.

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-beta1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.     

Reduced expression of the membrane skeleton protein beta1-spectrin (SPTBN1) is associated with worsened prognosis in pancreatic cancer

Spectrins are members of the superfamily of F-actin cross linking proteins that are important as scaffolding proteins for protein sorting, cell adhesion, and migration. In addition, spectrins have been implicated in TGF-beta signaling. The aim of the present study was to analyze the expression and localization of beta1-spectrin (SPTBN1) in pancreatic tissues. mRNA levels of SPTBN1 in cultured pancreatic cancer cell lines, as well as in normal pancreatic tissues (n=18), chronic pancreatitis (n=48) and pancreatic cancer tissues (n=66) were analyzed by real time quantitative RT-PCR. Localization of SPTBN1 in pancreatic tissues was determined by immunohistochemistry. SPTBN1 staining was assessed semi-quantitatively in 55 cancer tissues and survival analysis was carried out using the Kaplan-Meier method. Median SPTBN1 mRNA levels were 6.0-fold higher in pancreatic cancer tissues compared to the normal pancreas (p<0.0001) and 2.2-fold higher compared to chronic pancreatitis tissues (p=0.0002). In the normal pancreas, SPTBN1 was present in the cytoplasm of normal ductal cells and occasionally in pancreatic acinar and centroacinar cells. In pancreatic cancer tissues, SPTBN1 was present in the cytoplasm of pancreatic cancer cells. Low SPTBN1 protein expression indicated a tendency for worsened prognosis with a median survival of 14.0 months, versus 23.8 months for patients whose tumors expressed moderate/high levels of SPTBN1. In conclusion, reduced SPTBN1 expression correlated with shorter survival of pancreatic cancer patients, suggesting a tumor suppressor function of this gene, as has already been shown for other malignancies of the gastrointestinal tract.     

Interleukin-32 Expression in the Pancreas

Interleukin (IL)-32 is a recently described proinflammatory cytokine characterized by the induction of nuclear factor (NF)-κB activation. We studied IL-32 expression in human pancreatic tissue and pancreatic cancer cell lines. Tissue samples were obtained surgically. IL-32 expression was evaluated by standard immunohistochemical procedures. IL-32 mRNA expression was analyzed by Northern blotting and real time PCR analyses. IL-32 was weakly immunoexpressed by pancreatic duct cells. In the inflamed lesions of chronic pancreas, the ductal expression of IL-32 was markedly increased. A strong expression of IL-32α was detected in the pancreatic cancer cells. In pancreatic cancer cell lines (PANC-1, MIA PaCa-2, and BxPC-3 cells), the expression of IL-32 mRNA and protein was enhanced by IL-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. An inhibitor of phosphatidylinositol 3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) significantly suppressed the IL-1β-, IFN-γ- and TNF-α-induced IL-32 mRNA expression. The blockade of NF-κB and activated protein-1 activation markedly suppressed the IL-1β-, IFN-γ-, and/or TNF-α-induced IL-32 mRNA expression. Furthermore, IL-32-specific small interfering RNA significantly decreased the uptake of [³H]thymidine and increased the annexin V-positive population (apoptotic cells) in PANC-1 cells. IL-32 knockdown also suppressed the mRNA expression of antiapoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1). Pancreatic duct cells are the local source of IL-32, and IL-32 may play an important role in inflammatory responses and pancreatic cancer growth.     

The anti-apoptotic protein BAG-3 is overexpressed in pancreatic cancer and induced by heat stress in pancreatic cancer cell lines

Pancreatic cancer cells are usually resistant to apoptosis mediated by intrinsic or extrinsic factors. BAG-3 (Bis, CAIR), which was identified as a BAG-1-related protein, is a novel modulator of cellular anti-apoptotic activity that functions through its interaction with Bcl-2. In this study we analyzed BAG-3 expression in human pancreatic cancer tissues and cell lines. BAG-3 mRNA was expressed at moderate to high levels in all pancreatic cancer samples, but at low levels in normal pancreas tissues. In situ hybridization and immunohistochemistry analysis revealed that BAG-3 was present in the cancer cells within the pancreatic tumor mass. When BAG-3 mRNA was analyzed in other gastrointestinal cancers (hepatocellular carcinoma; esophageal, stomach and colon cancer), no difference was found from their corresponding normal controls. In pancreatic cancer cells, BAG-3 mRNA expression levels were strongly induced after heat stress, but not in response to members of the tumor necrosis factor (TNF)-alpha family (TNF-alpha, TRAIL, FasL). These findings indicate that in pancreatic cancer, in contrast to other gastrointestinal malignancies, increased levels of BAG-3 might function to block apoptosis. This characteristic of pancreatic cancer might contribute to its more aggressive growth behavior and poor responsiveness to treatment in vivo.     

Down-regulation of laminin-5 in breast carcinoma cells.

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.     

Aberrant gata-3 expression in human pancreatic cancer.

Gata-3 has been shown to specifically alter its expression patterns in different types of cancers. Recent evidence suggests that an interference of Gata-3 exists in the TGF-beta signaling pathway. To determine the role of Gata-3 in pancreatic cancer, pancreatic cancer samples were analyzed in comparison to normal pancreatic tissues. Furthermore, four different pancreatic cancer cell lines with different alterations of the TGF-beta pathway were studied. To evaluate if a potential relationship with TGF-beta signaling pathway exists, we correlated mRNA expression levels with the expression of TGF-betas, TGF-beta receptors, and Smad-3. Finally, we analyzed the influence of TGF-beta on Gata-3 expression in vitro. All pancreatic cancer samples demonstrated a marked overexpression of Gata-3 mRNA and protein. Immunohistochemical staining revealed strong and persistent cytoplasmic Gata-3 immunoreactivity in cancer cells. In an electrophoretic mobility shift assay, a disturbed nuclear translocation was confirmed. The expression of Gata-3 showed a significant correlation with the expression of TGF-betas, TGF-beta receptors, and Smad-3. TGF-beta responsive cell lines showed a downregulation of Gata-3 mRNA upon TGF-beta exposure, whereas in TGF-beta-unresponsive cell lines, Gata-3 mRNA expression persisted at high levels. Furthermore, strong specific upregulation of Gata-3 impaired nuclear translocation and its cooperative action with the TGF-beta pathway, suggesting that Gata-3 plays a central role in human pancreatic cancer.     

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.     

Bmi1 Enhances Tumorigenicity and Cancer Stem Cell Function in Pancreatic Adenocarcinoma

Background

Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.

Methods

We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.

Results

Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.

Conclusions

Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.     

Galectin-3 and Beclin1/Atg6 genes in human cancers: using cDNA tissue panel, qRT-PCR, and logistic regression model to identify cancer cell biomarkers

Background

Cancer biomarkers are sought to support cancer diagnosis, predict cancer patient response to treatment and survival. Identifying reliable biomarkers for predicting cancer treatment response needs understanding of all aspects of cancer cell death and survival. Galectin-3 and Beclin1 are involved in two coordinated pathways of programmed cell death, apoptosis and autophagy and are linked to necroptosis/necrosis. The aim of the study was to quantify galectin-3 and Beclin1 mRNA in human cancer tissue cDNA panels and determine their utility as biomarkers of cancer cell survival.

Methods and Results

A panel of 96 cDNAs from eight (8) different normal and cancer tissue types were used for quantitative real-time polymerase chain reaction (qRT-PCR) using ABI7900HT. Miner2.0, a web-based 4- and 3- parameter logistic regression software was used to derive individual well polymerase chain reaction efficiencies (E) and cycle threshold (Ct) values. Miner software derived formula was used to calculate mRNA levels and then fold changes. The ratios of cancer to normal tissue levels of galectin-3 and Beclin1 were calculated (using the mean for each tissue type). Relative mRNA expressions for galectin-3 were higher than for Beclin1 in all tissue (normal and cancer) types. In cancer tissues, breast, kidney, thyroid and prostate had the highest galectin-3 mRNA levels compared to normal tissues. High levels of Beclin1 mRNA levels were in liver and prostate cancers when compared to normal tissues. Breast, kidney and thyroid cancers had high galectin-3 levels and low Beclin1 levels.

Conclusion

Galectin-3 expression patterns in normal and cancer tissues support its reported roles in human cancer. Beclin1 expression pattern supports its roles in cancer cell survival and in treatment response. qRT-PCR analysis method used may enable high throughput studies to generate molecular biomarker sets for diagnosis and predicting cancer treatment response.     

Differential Cellular Localization of Galectin-1 and Galectin-3 in the Regressing Corpus Luteum of Mice and Their Possible Contribution to Luteal Cell Elimination

Galectin-1 and galectin-3, β-galactoside–binding lectins, are predominantly expressed in the regressing corpus luteum (CL) of mouse ovary. This study revealed the expression patterns and cellular localizations of galectins during CL formation and regression by ISH and IHC. Galectin-1 mRNA expression temporarily increased in active CL, preceding the expression of progesterone degradation enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD), which represents functional luteolysis. The expressions of both galectin-1 and galectin-3 remarkably increased in the structurally regressing CL, which vigorously expressed 20α-HSD and contained abundant apoptotic luteal cells. Ultrastructurally, galectin-1– and galectin-3–immunoreactive cells were identified as fibroblasts and infiltrating macrophages, respectively. In addition, some populations of luteal cells themselves expressed galectin-3 in regressing CL and formed unique demarcation membranes in the cytoplasm, showing a non-typical apoptotic feature. Ovary of adult mice with repeated estrus cycles contained CL of three different generations. Among them, the old CL formed during previous estrus cycles consisted of galectin-3–positive luteal cells. The galectin-3–positive old CL was resistant to apoptosis and seemed to be eliminated by a mechanism different from apoptosis. The stage- and cell-specific expression of galectin in CL suggests its differential contribution to luteolysis, and this expression may be mediated by major regulatory molecules of CL function, prolactin and/or prostaglandin F2α. (J Histochem Cytochem 58:741–749, 2010)     

Localization of mRNAs and Proteins in Methyl Methacrylate�Cembedded Tissues

Precise localization of proteins and mRNA in histological sections is necessary for evaluating spatial gene expression patterns. Here we report sensitive detection of the gene products in fish tissues by immunohistochemistry (IHC) and in situ hybridization (ISH) assays on sections of whole specimens and vertebra embedded in methyl methacrylate (MMA) resin. This plastic resin favors easy preparation of various specimen types and enables preparation of large sections with well-preserved cell morphology. IHC analysis of the muscle regulatory factor MyoD in transverse sections of juvenile cod revealed MyoD-positive cells in the dorsolateral parts of the adaxial muscle. ISH revealed less spatially restricted signals of the bone morphogenic protein bmp4 in muscle and brain. To assess the applicability of ISH on sections of bony tissue, col1a1 and col2a1 expression was investigated in non-decalcified vertebra sections of Atlantic salmon. The former was identified in both chondrocytes and osteoblasts, whereas the latter was mostly evident in chondrocytes. We conclude that MMA resin offers easy preparation of large and problematic tissues and the possibility of carrying out both IHC and ISH analyses using standard protocols. (J Histochem Cytochem 57:825–830, 2009)