An antibody to de-N-acetyl sialic acid containing-polysialic acid identifies an intracellular antigen and induces apoptosis in human cancer cell lines

Polysialic acid (PSA), an α2,8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac), is developmentally regulated and its expression is thought to be restricted to a few tissues in adults. Recently, we showed that two human pathogens expressed a derivative of PSA containing de-N-acetyl sialic acid residues (NeuPSA). Here we show that an epitope identified by the anti-NeuPSA monoclonal antibody, SEAM 3 (SEAM 3-reactive antigen or S3RA), is expressed in human melanomas, and also intracellularly in a human melanoma cell line (SK-MEL-28), a human T cell leukemia cell line (Jurkat), and two neuroblastoma cell lines (CHP-134 and SH-SY5Y). SEAM 3 binding induced apoptosis in the four cell lines tested. The unusual intracellular distribution of S3RA was similar to that described for the PSA polysialyltransferases, STX and PST, which are also expressed in the four cell lines used here. Interestingly, suppression of PST mRNA expression by transfection of SK-MEL-28 cells with PST-specific short interfering RNA (siRNA) resulted in decreased SEAM 3 binding. The results suggest further studies of the utility of antibodies such as SEAM 3 as therapeutic agents for certain malignancies.     

Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

ABSTRACT. Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly‐α2,8 N‐acetyl neuraminic acid (PSA) and PSA containing de‐N‐acetyl neuraminic acid (NeuPSA). Expression of rosette‐specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent‐insoluble cell fraction with both PSA and NeuPSA‐reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.     

High-throughput transcriptomic and RNAi analysis identifies AIM1, ERGIC1, TMED3 and TPX2 as potential drug targets in prostate cancer

Prostate cancer is a heterogeneous group of diseases and there is a need for more efficient and targeted methods of treatment. In this study, the potential of gene expression data and RNA interference technique were combined to advance future personalized prostate cancer therapeutics. To distinguish the most promising in vivo prevalidated prostate cancer drug targets, a bioinformatic analysis was carried out using genome-wide gene expression data from 9873 human tissue samples. In total, 295 genes were selected for further functional studies in cultured prostate cancer cells due to their high mRNA expression in prostate, prostate cancer or in metastatic prostate cancer samples. Second, RNAi based cell viability assay was performed in VCaP and LNCaP prostate cancer cells. Based on the siRNA results, gene expression patterns in human tissues and novelty, endoplasmic reticulum function associated targets AIM1, ERGIC1 and TMED3, as well as mitosis regulating TPX2 were selected for further validation. AIM1, ERGIC1, and TPX2 were shown to be highly expressed especially in prostate cancer tissues, and high mRNA expression of ERGIC1 and TMED3 associated with AR and ERG oncogene expression. ERGIC1 silencing specifically regulated the proliferation of ERG oncogene positive prostate cancer cells and inhibited ERG mRNA expression in these cells, indicating that it is a potent drug target in ERG positive subgroup of prostate cancers. TPX2 expression associated with PSA failure and TPX2 silencing reduced PSA expression, indicating that TPX2 regulates androgen receptor mediated signaling. In conclusion, the combinatorial usage of microarray and RNAi techniques yielded in a large number of potential novel biomarkers and therapeutic targets, for future development of targeted and personalized approaches for prostate cancer management.     

Expression of annexin VI (p68, 67 kDa-calelectrin) in normal human tissues: evidence for developmental regulation in B- and T-lymphocytes

This paper describes the tissue distribution of annexin VI, a Ca²⁺-dependent phospholipid binding protein, and a member of the annexin super-gene family. In order to determine whether annexin VI expression correlated with a particular functional phenotype, an extensive series of non-pathological human tissues were examined, in which annexin VI was detected either immunohistochemically or by immunofluorescence, using a rabbit polyclonal anti-(human annexin VI)-IgG of known specificity. Although most tissues investigated were found to express annexin VI, the protein was usually confined to highly specific cell types within each tissue, the staining generally appearing cytoplasmic and diffuse. There was particularly good correlation between annexin VI expression and hormone secreting cells, with positive staining in the islet cells of the pancreas, the Leydig cells of the testis and the cells of the adrenal cortex. A notable exception was the parathyroid gland, which lacked detectable annexin VI. Although the protein was absent in most epithelia, it was expressed strongly in certain secretory epithelia; e.g. the ductal epithelial cells of the salivary glands and non-lactating breast, and the sweat glands and their ducts. The observation that the epithelial cells of lactating breast failed to stain for annexin VI suggests functional regulation of protein expression in this tissue. However, the most interesting finding was that annexin VI expression appeared to be developmentally regulated in B- and T-lymphocyte differentation, with negative staining in the proliferating B cells of the germinal centre of the lymph nodes, but strong staining in the mature small lymphocytes of the cortex, mantle zone and paracortex.     

Expression of annexin VI (p68, 67 kDa-calelectrin) in normal human tissues: evidence for developmental regulation in B- and T-lymphocytes.

This paper describes the tissue distribution of annexin VI, a Ca(2+)-dependent phospholipid binding protein, and a member of the annexin super-gene family. In order to determine whether annexin VI expression correlated with a particular functional phenotype, an extensive series of non-pathological human tissues were examined, in which annexin VI was detected either immunohistochemically or by immunofluorescence, using a rabbit polyclonal anti(human annexin VI)-IgG of known specificity. Although most tissues investigated were found to express annexin VI, the protein was usually confined to highly specific cell types within each tissue, the staining generally appearing cytoplasmic and diffuse. There was particularly good correlation between annexin VI expression and hormone secreting cells, with positive staining in the islet cells of the pancreas, the Leydig cells of the testis and the cells of the adrenal cortex. A notable exception was the parathyroid gland, which lacked detectable annexin VI. Although the protein was absent in most epithelia, it was expressed strongly in certain secretory epithelia; e.g. the ductal epithelial cells of the salivary glands and non-lactating breast, and the sweat glands and their ducts. The observation that the epithelial cells of lactating breast failed to stain for annexin VI suggests functional regulation of protein expression in this tissue. However, the most interesting finding was that annexin VI expression appeared to be developmentally regulated in B- and T-lymphocyte differentiation, with negative staining in the proliferating B cells of the germinal centre of the lymph nodes, but strong staining in the mature small lymphocytes of the cortex, mantle zone and paracortex.     

Claudin-16 reduces the aggressive behavior of human breast cancer cells

Claudin-16 (Paracellin-1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re-absorption of magnesium and calcium in the kidney. This study examined expression of Claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin-16 levels and patient prognosis. Insertion of the Claudin-16 gene into MDA-MB-231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and "tighter" colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno-stained. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, normalized with cytokeratin-19 levels). Levels of expression of Claudin-16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin-16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type.     

Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis

Rat monoclonal antibodies (MAb) directed to mouse Ig heavy and light chain determinants were produced. A rat anti-mouse light chain MAb (RAMOL-1) which bound to all (24/24) mouse Ig of the kappa light chain type and with varying strength to 4/4 lambda light chain-bearing Ig was evaluated as a general secondary reagent, together with two MAb that bound to the heavy chain of mouse IgG. They were conjugated with biotin or FITC and used in immunohistochemical and immunofluorescence assays to detect mouse monoclonal antibodies binding to antigens expressed in rat and human tissues and cells. As compared to commercially available polyclonal reagents, RAMOL-1 gave higher staining contrast by showing lower background staining and equal or higher staining of the primary MAb tested. This was a result of two main effects. First, crossreactivity with endogenous Ig and tissue type-specific determinants was eliminated. With polyclonal anti-mouse Ig reagents, binding to endogenous Ig was noted in vascular spaces and on Ig-bearing cells, and to rat gastric mucosa and epithelial tumor tissue in frozen tissue sections, even when diluted in high concentrations of serum homologous to the tissue. Second, binding of the secondary reagent was reduced to cells and tissues prone to have high nonspecific binding capability, such as monocytes/macrophages and formalin-fixed, paraffin-embedded tissue. Owing to unlimited and reproducible access to this homogeneous reagent, RAMOL-1 is used as second antibody to standardize the procedure used for immunohistochemical grading of human malignant tumors by determination of blood group antigen expression detected with mouse MAb.     

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

Discovery of targeting peptides for selective therapy of medullary thyroid carcinoma

BACKGROUND: Adenovirus efficiently infects a broad range of target cells, thereby preventing selective gene transfer. Moreover, several cell types and tissues including primary tumors are refractory to adenoviral infection, mainly because of low expression levels of coxsackie-adenovirus receptor (CAR). Thus, identification of cancer-selective ligands which yield gene transfer to neoplastic cells by minimizing transduction of normal cells is a key issue for successful cancer therapy. METHODS: We initially analyzed adenoviral receptor expression in human medullary thyroid carcinoma (MTC) cells. MTC cell-specific peptides were isolated by biopanning a phage display peptide library on cultured cancer cells and on tumors in vivo and further characterized. RESULTS: We found significant differences in CAR and alphav-integrin protein levels between MTC-derived TT cells in vitro and established xenograft tumors in mice, indicating a lack of alphav-integrin expression on growing tumors. MTC-specific candidates were identified by performing three rounds of subtraction. Selected phages showed up to 22-fold higher binding efficiency for TT cells when compared with wild-type M13 phage or other human cell lines and tumor tissue in vivo. Homing to TT cells of the best binding phage was clearly blocked in the presence of specific peptide, whereas no phage competition was observed with an unspecific peptide. The best binding peptide mediated efficient internalization of the phage. Importantly, specific binding and internalization was also mediated by the identified peptide within the adenoviral context. CONCLUSIONS: Our results indicate that the identified ligand should be suitable to improve selectivity of adenoviral gene transfer to medullary thyroid tumors in vivo.     

Immunohistochemical analysis of human lung cancers with anti-ras p21 monoclonal antibodies

The expression of ras oncogene product p21 in human malignant pleurisy and primary lung cancer was studied immunocyto-histochemically with monoclonal antibodies (MoAbs) rp-28 and rp-35 against ras p21. In pleural effusion cells, cancer cells revealed more intensively positive reaction with MoAb rp-35 than with MoAb rp-28, especially in the plasma membrane, and no positive reaction was obtained in any kind of inflammation cells with the exception of faintly positive reaction in the cytoplasm of macrophages. In primary lung cancers, well or moderately differentiated adenocarcinoma tissues showed higher reactivity with MoAb rp-28 than those of poorly differentiated adenocarcinoma or any other histological subtype of lung cancer. With MoAb rp-35, intensively positive reaction was obtained in most of cases with all different histological subtypes of lung cancer. The staining in cancer cells was usually localized intensively to the plasma membrane and weakly to the cytoplasm with both MoAbs. Normal bronchial epithelial and glandular tissues showed only cytoplasmic staining. These two MoAbs, especially MoAb rp-35, may be useful in clinicopathological applications for the diagnosis of malignant pleurisy and primary lung cancer.     

Effects of long-term androgen administration on breast tissue of female-to-male transsexuals.

Our aim was to examine the effects of androgen administration on breast tissue histology of female-to-male transsexuals and to study the immunohistochemical expression of three human tissue kallikreins, hK3 (PSA), hK6, and hK10. We studied 23 female-to-male transsexuals who were treated with injectable testosterone for 18-24 months. We also used 10 control female breast tissues. All tissues were fixed in buffered formalin, embedded in paraffin, and examined by hematoxylin-eosin staining and immunohistochemical staining for PSA, hK6, and hK10. Females treated with androgens exhibited similar involutionary changes as those seen in breast of menopausal women, such as marked reduction of glandular tissue, involution of the lobuloalveolar structures, and prominence of fibrous connective tissue, but presence of only small amounts of fat tissue. Fibrocystic lesions were generally not observed. In immunohistochemistry, in control breast tissues, we found moderate to strong cytoplasmic immunoexpression of hK6 and hK10 in the epithelial ductal and lobuloalveolar structures, but myoepithelial cells were negative. Luminal secretions were also positive. In menopausal breast, the immunoexpression of hK6 and hK10 was weaker and focal. No control case showed immunoexpression for PSA. In female-to-male transsexuals, one case showed focal PSA cytoplasmic immunoexpression in the epithelium of moderately involuting lobules. Long-term administration of androgens in female-to-male transsexuals causes marked reduction of glandular tissue and prominence of fibrous connective tissue. These changes are similar to those observed at the end-stage of menopausal mammary involution.     

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.     

Widespread expression of γ-glutamyl cyclotransferase suggests it is not a general tumor marker

γ-Glutamyl cyclotransferase (GGCT) contributes to the γ-glutamyl cycle that regulates glutathione metabolism. Although GGCT has been implicated in several studies as a possible cancer marker, little is known about its distribution in cells and tissues. The authors investigated GGCT expression in normal tissues and tumors using Western blots and immunohistochemistry with a novel anti-GGCT monoclonal antibody. GGCT was detected in most organs and was mainly found in epithelial cells. Although the intracellular distribution was mainly cytoplasmic, in some situations, nuclear staining was strong. A significant increase in the expression of GGCT was found in tumors of the lung, esophagus, stomach, bile duct, and uterine cervix. In contrast, there was a significant decrease in expression in renal and urothelial tumors. These results suggest that GGCT may be a biomarker of tumors in a limited range of organs.     

microRNA-21在食管癌细胞增殖、迁移和凋亡中的作用

本研究检测了40例食管癌组织和40例癌旁组织中的miR-21、PTEN、PI3K和AKT表达,并通过转染miR-21抑制剂来敲低人食管癌细胞系EC9706的miR-21表达,考察了miR-21对食管癌细胞生长的影响。研究发现,食管癌组织中PTEN蛋白的阳性染色评分低于癌旁组织(p<0.05),而PI3K和AKT蛋白的阳性染色评分高于癌旁组织(p<0.05)。miR-21在人食管癌组织中被上调(3.56 vs 1.21,p<0.05)。转染miR-21抑制剂导致PTEN蛋白表达升高,而PI3K和AKT蛋白表达降低(p<0.05)。转染miR-21抑制剂抑制了EC9706细胞的增殖和迁移,但促进了细胞凋亡(p<0.05)。miR-21的上调可通过激活PTEN/PI3K/AKT信号通路来促进食道癌细胞的增殖和迁移,并抑制细胞凋亡。     

High SEPT9_i1 Protein Expression Is Associated with High-Grade Prostate Cancers

Septins are a family of GTP-binding cytoskeleton proteins expressed in many solid tumors. Septin 9 (SEPT9) in particular was found overexpressed in diverse carcinomas. Herein, we studied the expression of SEPT9 isoform 1 protein (SEPT9_i1) in human prostate cancer specimens. We utilized immunohistochemical staining to study the expression of SEPT9_i1 protein. Staining level was analyzed in association with clinical characteristics and the pathological Gleason grade and score. Fifty human prostate cancer specimens (42 primary tumors and 8 metastatic lesions) were stained by SEPT9_i1 antibody and analyzed. SEPT9_i1 protein was expressed in prostate cancer cells but absent in normal epithelial cells. The intensity of staining was correlated proportionally to pretreatment prostate-specific antigen (PSA) blood levels and Gleason score (P < 0.05). SEPT9_i1 was highly expressed in all metastatic lesions. A significant assocation between SEPT9_i1 expression and high Gleason score on multivariate linear regression analysis was found. We conclude that SEPT9_i1 is expressed in high-grade prostate tumors suggesting it has a significant role in prostate tumorigenesis and that it could serve as a molecular marker for prostate tumor progression.     

Immunohistochemical detection of cytoplasmic LC3 puncta in human cancer specimens

Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.     

A multiple marker analysis of apoptosis-associated protein expression in non-small cell lung cancer in a Chinese population

A failure to undergo apoptosis is widely thought to be an important event in cancer formation and progression. Although there have been many studies in vitro that provide evidence for this suggestion, the roles of apoptosis-associated proteins in cancer tissues in vivo are not as yet fully understood. Moreover, multiple marker analyses of apoptosis-associated protein expression in non-small cell lung cancer (NSCLC) tissues are scarce. In the present study, we investigate the expression of a group of apoptosis-associated proteins including bcl-2, caspase-3, fas, fas ligand (fasL) and survivin, and its clinical significance in NSCLC tissues using immunohistochemistry (IHC). Bcl-2 staining in cancer tissue cells was found in cytoplasm and the positive rate was 38.2% (29/76). Caspase-3 staining was mainly seen in cytoplasm of cancer tissue cells (53.9% [41/76]) with a few cases of nuclear staining (6.6% [5/76]). Fas staining was seen in cytomembrane (15.8% [12/76]) and cytoplasm (42.1% [32/76]) of cancer tissue cells. Likewise, fasL also showed staining in cytoplasm (55.3% [42/76]) and cytomembrane (44.7% [34/76]) of cancer tissue cells. Survivin staining was seen in cytoplasm but not nuclear of cancer tissue cells and the positive rate was 48.7% (37/76). Higher cytoplasm expression of bcl-2 was associated with large tumor size (≥ 3 cm) in NSCLC (p < 0.05). Decreased cytoplasm expression of fas was associated with poor grade in NSCLC (p < 0.05). A negative correlation was found between bcl-2 and cytoplasm caspase-3 expression in NSCLC (p < 0.001). No separate expression of the apoptosis-associated proteins in NSCLC was linked to overall survival of patients (p > 0.05). Multiple marker analyses revealed caspase-3+/cytomembrane fasL- to be linked to better survival of patients with NSCLC (p < 0.05). These results indicate that apoptosis- -associated proteins may impact a variety of clinicopathological features of NSCLC and may co-operatively influence the prognosis of patients with this malignant tumor.