Positive mRNA Signals Research Articles

HER2 as marker for the detection of circulating tumor cells

In breast cancer patients, the onset of subclinical tumor cell spread is the putative precursor stage of solid metastases and can be assessed with the detection of disseminated tumor cells (DTC) in bone marrow samples or circulating tumor cells (CTC) in the blood by immunocytochemical and molecular techniques. Sequential peripheral blood analyses should be more acceptable than bone marrow aspirations and many research groups are currently assessing CTC with different methods. In patients with no clinical signs of overt metastases, detection rates range from 10 to 100% [1], (Table 1). The detection of CTC is of potential clinical relevance in the context of a growing number of therapeutic options, especially in the adjuvant setting when no tumor is present. In contrast to patients with metastatic disease, less information about the prognostic relevance of CTC in the blood of patients with earlystage disease is available [2]. In this context, the study published by Apostolaki and coworkers in this issue of Breast Cancer Research and Treatment is of importance [3]. The authors use a nested RT-PCR assay for HER2 mRNA to detect CTC in patients with primary breast cancer after surgery and before the start of systemic treatment. With this method, they detect CTC in 24.5% of 216 patients. The detection of a positive mRNA signal for HER2 is an independent prognostic factor for disease free and overall survival. No correlation of CTC detection by HER2 mRNA and HER2 status of the primary tumors was observed. In metastatic patients, it has been demonstrated that patients with HER2 negative primary tumors show HER2 positive CTC. Moreover, a subset of these patients were treated with trastuzumab and showed clinical response [4, 5]. Since the number of patients included in these studies were small, no conclusive data have been obtained. The intriguing perspective of this study is on one hand the potential clinical impact since HER2 is a prominent therapeutic target in breast cancer and on the other hand the biologic hypothesis supported by this study: HER2-positive cells are more frequently disseminated from the primary tumors. When looking at the seemingly high frequency of HER-positive CTC here one has to bear in mind that HER2 is usually not overexpressed or amplified in all cells of a primary tumor, even in those tested as ‘‘positive’’. In addition, the applied RNA-based test is not able to detect a heterogeneity in HER2 expression between different CTC (i.e., the concomitant presence of HER2-positive and HER2-negative CTC in one sample), which was, however, observed by the authors of this study when performing a immunocytochemical or FISH analysis in a small subset of patients tested HER2 positive by RNA.

Transcriptome Analysis of a cDNA Library from Adult Human Epididymis

Mammalian Gene Collection (MGC) verified over 9000 human full-ORF genes and FLJ Program reported 21 243 cDNAs of which 14 409 were unique ones and 5416 seemed to be protein-coding. The pity is that epididymis cDNA library was missing in their sequencing target list. Epididymis is a very important male accessory sex organ for sperm maturation and storage. Fully differentiated spermatozoa left from testis acquire their motility and capacity for fertilization via interactions with the epididymal epithelium duct lumen during passage through this convoluted duct. Here, we report that 20 000 clones from a healthy male epididymis cDNA library have been sequenced. The sequencing data provided 8234 known sequences and 650 unknown cDNA fragments. Hundred and six of 650 unknown cDNA clone inserts were randomly selected for fully sequencing. There were 25 unknown unique sequences and 19 released but unreported sequences came out. By northern blot analysis, four sequences randomly selected from the 19 released sequences with no known function showed positive mRNA signals in epididymis and testis. The signals for three of six from those unknown group showed as epididymis abundant in a region-specific manner but not in the testis and other tissues tested. All the sequencing data will be available on the website www.sdscli.com.

Systematic expression profiling of the gastric H+/K+ ATPase in human tissue

Objective. The potassium-competitive acid blockers (P-CABs), comprise a new, innovative group of competitive and reversible inhibitors of the gastric H+/K+ ATPase. Our aim was to identify sites of expression of the H+/K+ ATPase that are potential targets of these compounds by examining the expression profile of the gastric H+/K+ ATPase in the human body from a broad range of tissues. Material and methods. Expression profiling was done by quantitative mRNA analysis (TaqMan PCR). Tissues that were mRNA-positive for the alpha subunit were investigated further by Western blot and immunohistochemistry (IHC) for the presence of gastric H+/K+ ATPase protein. Results. In addition to the very high expression levels in the stomach, the adrenal gland, cerebellum and pancreas gave unexpectedly positive mRNA signals for the alpha subunit of gastric H+/K+ ATPase. However, they were negative for mRNA of the beta subunit, and Western blot and IHC were negative for alpha and beta subunit protein. Another group of tissues with low alpha subunit mRNA expression including the frontal cortex, cortex grey matter, testis, thymus and larynx submucosa were also found negative for both alpha and beta subunit protein. In contrast to mouse kidney, no gastric H+/K+ ATPase could be detected in human kidney. Conclusions. We therefore conclude that the only organ in humans expressing significant levels of the P-CAB target gastric H+/K+ ATPase is the stomach.

Prognostic impact of cysteine proteases cathepsin B and cathepsin L in pancreatic adenocarcinoma.

The cysteine proteases cathepsin B (CTSB) and L (CTSL) have been implicated in tumor spread and metastatic formation. In pancreatic adenocarcinoma, the role of these proteases is not very well defined. To find out which cell types produce CTSB and CTSL and to evaluate the prognostic impact of these proteases, 70 specimens from curatively resected patients with pancreatic adenocarcinoma were examined by in situ hybridization and immunohisto-chemistry. Seventy patients with ductal adenocarcinoma of the pancreas were studied after R0 resection with a follow-up of at least 3 years. CTSB and CTSL expression was performed immunohisto-chemically using polyclonal anti-CTSB and CTSL antibodies. To detect cell types involved in producing CTSB and CTSL as well as the intracellular localization of specific mRNA sequences, nonisotopic in situ hybridization was performed. The correlations among CTSB and CTSL expression, clinicopathologic parameters, and clinical outcome were analyzed. The immunoreactivity was 96% for CTSB and 90% for CTSL. Positive mRNA signals were obtained in the cytoplasm tumor cells, macrophages, and fibroblasts in 77% for CTSB and 81% for CTSL, respectively. Statistical analysis showed a significant correlation between CTSB/CTSL expression and tumor grading (P < 0.05) and between CTSB and lymphatic invasion (P = 0.05). Kaplan-Meier analyses revealed statistical significance for CTSB/CTSL expression with the survival after curative resection (P < 0.05). Both proteases are strong prognostic markers in multivariate analysis (P = 0.0001) beside UICC stage, nodal status, tumor size, and grading (P < 0.05). Furthermore, CTSB expression is an independent prognostic marker for cancer recurrence within 6 months after curative surgery in multivariate analysis (P = 0.0001). CTSB and CTSL are strong and independent prognostic markers in resectable pancreatic adenocarcinoma rather than UICC stage, TNM classification, or tumor grading. Furthermore, CTSB is a predictor for early recurrence after curative resection. These data underline the significance of tumor-associated proteolysis for cancer invasion and metastasis and may lead to defining subgroups of patients with early recurrence and poor outcome.

Relationship of messenger RNA reverse transcriptase-polymerase chain reaction signal to Campylobacter spp. viability.

SUMMARY. Discriminating viable from dead cells is of importance in the development of bacterial detection methods. A positive reverse transcriptase-polymerase chain reaction (RT-PCR) amplification signal was tested as a potential predictor of chick colonization. Some researchers have suggested that the presence of messenger RNA (mRNA) may not correlate with cell viability. Chicken colonization by cells that have positive mRNA signal but that are noncultivable would provide a correlation in cell viability and persistence of mRNA. The role of a viable but noncultivable (VBNC) form of Campylobacter spp. for colonization of poultry could be verified by such an mRNA signal. The levels of four strains of Campylobacter spp., previously isolated from poultry feces, declined progressively over time, and loss of cultivability occurred after 6 to 7 wk incubation in phosphate-buffered saline (PBS) at 4 C. Cold-stored, noncultivable and heat-inactivated (60 C for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay, depending on the target transcripts and strains used, although all fresh cultures showed mRNA signals. For the most part, signals of mRNA species from VBNC and heat-killed Campylobacter spp. AH-1, AH-2, and CH-3 persisted. RT-PCR amplification of transcripts originating from the tkt and cmp genes and a 256-base pair amplicon (from a previously described putative haem-copper oxidase) provided consistent signals, whereas transcripts from the flaA gene did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not cultivable by conventional culture-based methods, did not establish colonization in the intestine of chicks 7 days after challenge. These results lead us to question the correlation between mRNA durability with cell viability as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.

High expression of vascular endothelial growth factor predicts early recurrence and poor prognosis after curative resection for ductal adenocarcinoma of the pancreas.

Only curative resection for pancreatic adenocarcinoma is related to a favorable prognosis, but the overall survival after surgery still remains poor, and early recurrence is frequently observed. Because recurrence is the limiting factor and the main cause of death after curative resection, the identification of markers that predict early postoperative recurrence is of paramount importance. Angiogenesis is essential for tumor growth and metastases; therefore, we set out to clarify whether vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) correlate with early recurrence and poor prognosis after curative resection. A second goal was to characterize the VEGF-producing cells and the subcellular distribution. Seventy patients with ductal adenocarcinoma of the pancreas were studied after curative resection with a follow-up of at least 2 years. The MVD quantification was performed immunohistochemically with use of a monoclonal antibody to CD34. The VEGF expression was studied with use of polyclonal antibody. To detect the intracellular localization of specific VEGF mRNA sequences, nonisotopic in situ hybridization was performed. The correlations among VEGF expression and MVD, clinicopathologic parameters, and clinical outcome were then statistically analyzed. The VEGF immunoreactivity was 88.6%, and positive mRNA signals were obtained in the cytoplasm of carcinoma and endothelial cells in 81.4%. Furthermore, we observed tumor-associated macrophages close to infiltrating carcinoma cells. All endothelial cells showed positive immunoreactivity to the anti-CD34 antibody, and a median distribution of 85 vessels/x200 field was observed. A significant correlation (p < 0.05) was found between the MVD and the International Union Against Cancer (UICC) stage. Statistical analysis showed a significant correlation between VEGF expression and the height of MVD (p < 0.05). Kaplan-Meier analyses revealed that VEGF expression and MVD had a statistically significant correlation with survival after curative resection (p < 0.05). Furthermore, multivariate analysis indicated that VEGF expression is an independent prognostic marker for cancer recurrence within 8 months after curative surgery (p = 0.003). In pancreatic adenocarcinoma, the VEGF expression and the height of MVD are closely correlated, and both-rather than UICC stage and TNM classification (tumor size and nodal involvement)-are markers of prognostic relevance after curative resection. Furthermore, VEGF is a predictor of early recurrence after curative resection. The current study indicates that VEGF may promote the distribution of metastases, leading to early cancer recurrence and poor outcome.

Evidence of leptin expression in normal and polycystic human ovaries.

Leptin, the 'obese' protein, is found in cultured granulosa cells derived from human pre-ovulatory follicles. However, the occurrence of leptin has not been studied in intact ovaries, either normal or polycystic, until now. Paraffin sections from 25 human ovaries of different cycle stages and 25 wedge resections of polycystic ovaries were investigated by means of immunochemistry. Additionally, three ovaries were available for reverse transcription-polymerase chain reaction analysis. Leptin-positive cells were located in the granulosa cells of pre-antral follicles, and distinctly in the thecal layer of intact and regressing antral follicles. In the corpus luteum (CL) in the developmental stage, the former epithelioid leptin-positive thecal cells became fibroblast-like in the septum. In the CL of the secretory stage, single leptin-positive cells were detected between luteal cells. In polycystic ovaries, leptin-positive cells were noted both in the hypertrophied thecal layer and in the luteinized granulosa layer. Our findings on leptin expression at the protein level were confirmed by a positive mRNA signal for leptin in granulosa cells and in the CL. Additionally, mRNA of the full-length leptin receptor OB-R and of the short isoforms B219.1-B219.3 was identified in granulosa cells and the CL, as well as in the cortex and medulla. We conclude that leptin is produced in the ovary and may act in autocrine and paracrine ways.

Inhibins in the male Göttingen miniature pig: Leydig cells are the predominant source of inhibin B.

The expression of inhibin subunits in the testes of the Göttingen miniature pig was examined by in situ hybridization and immunohistochemistry. In addition, the major forms were determined by enzyme-linked immunosorbent assay (ELISA). Strong positive immunostaining for the inhibin alpha subunit was observed in Sertoli and late-stage germ cells, but it was weak in Leydig cells. However, Leydig cells showed strong positive staining for the betaA subunit, but Sertoli cells and spermatogonia showed a weak reaction. Strong positive immunostaining for the betaB subunit was observed in Leydig cells but spermatogonia showed weak staining for it. In contrast to the staining specificity of inhibin alpha and betaA subunits, the betaB subunit did not exhibit positive staining in Sertoli cells. In situ hybridization revealed that although the a subunit mRNA signal was highly expressed in all cell types, the reaction appeared to be stronger in Sertoli cells and spermatogonia than in Leydig cells. betaA subunit mRNA expression was somewhat identical to that of the alpha subunit, however, germ cells showed a weak stain for it. A strong, positive mRNA signal for the betaB subunit was confined to Leydig cells and late-stage germ cells. ELISA results showed that concentrations of inhibin B and inhibin pro-alphaC were high in the circulation and testes. In contrast, inhibin A levels in both plasma and testes were undetectable. The present results strongly suggest that inhibin B is the major form of circulating inhibin and that Leydig cells are the predominant source of this dimeric inhibin in male Göttingen miniature pigs. Furthermore, the germ cells also appear to be an important source of circulating inhibins.

Mutual Induction of TGFβ1 and NGF after Treatment with NGF or TGFβ1 in Grafted Chromaffin Cells of the Adrenal Medulla

Chromaffin cells have been recognized for their ability to transform into sympathetic ganglion-like cells in response to nerve growth factor (NGF) or to stimulation of other neurotrophic factors. Transforming growth factor β (TGFβ) family members have been shown to potentiate the effect of different trophic factors. The aim of this study was to investigate if TGFβ may influence NGF-induced neuronal transformation and regulation of NGF, TGFβ1, and their receptors in the adult rat chromaffin tissue after grafting. Intraocular transplantation of adult chromaffin tissue was employed and grafts were treated with TGFβ1 and/or NGF. Graft survival time was 18 days after which the grafts were processed for TGFβ luciferase detection assay, NGF enzyme immunoassay, or in situ hybridization. In grafts stimulated with NGF, increased levels of TGFβ1 and TGFβ1 mRNA were detected. When grafts instead were treated with TGFβ1, enhanced levels of NGF protein were found. Furthermore, a positive mRNA signal corresponding to the transforming growth factor II receptor (TβRII) was found in the chromaffin cells of the normal adrenal medulla as well as after grafting. No increase of TβRII mRNA levels was detected after transplantation or after TGFβ1 treatment. Instead a reduction of TβRII mRNA expression was noted after NGF treatment. NGF stimulation of grafts increased the message for NGF receptors p75 and trkA in the chromaffin transplants. Grafts processed for evaluations of neurite outgrowth were allowed to survive for 28 days and were injected weekly with NGF and/or TGFβ1. NGF treatment resulted in a robust innervation of the host irides. TGFβ1 had no additive effect on nerve fiber formation when combined with NGF. Combined treatment of NGF and anti-TGFβ1 resulted in a significantly larger area of reinnervation. In conclusion, it was found that NGF and TGFβ1 may regulate the expression of each other's protein in adult chromaffin grafts. Furthermore, TβRII mRNA was present in the adult rat chromaffin cells and became downregulated as a result of NGF stimulation. Although no synergistic effects of TGFβ1 were found on NGF-induced neurite outgrowth, it was found that TGFβ1 and NGF signaling are closely linked in the chromaffin cells of the adrenal medulla.

An in situ hybridization histochemistry method for the use of alkaline phosphatase-labeled oligonucleotide probes in small intestine.

We have developed a method of non-radioactive in situ hybridization histochemistry using alkaline phosphatase-labeled oligonucleotide probes to detect gene expression in the intestine. Because the intestine contains a large amount of endogenous alkaline phosphatase activity, mild acid pretreatment of the tissue sections was required to inactivate the alkaline phosphatase. Acid pre-treatment dramatically reduced the endogenous activity without affecting the efficiency of hybridization or the probe's ability to reveal a positive mRNA signal. Furthermore, the addition of polyvinyl alcohol to the substrate solution helped to keep the background staining low without adversely affecting the intensity of the signal. The current protocol allows rapid and sensitive detection of sites of gene expression in intestinal tissue.