DEVELOPMENT OF HUMAN KIDNEY AS SHOWN BY MICRODISSECTION. I. PREPARATION OF TISSUE WITH REASONS FOR POSSIBLE MISINTERPRETATION OF OBSERVATIONS.
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Dissection↗
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Reverse transcription-polymerase chain reaction-based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sections.
Quantitative differences in the expression of oncogenes are a critical feature of the cancer process. Several methods are currently available for assessing differential gene expression, but none can be used to determine quantitative changes in gene expression from small numbers of cells. The ability to conduct this type of quantitative analysis would be useful in the study of definable, early stages of carcinogenesis when very few cells are involved. We therefore developed a highly sensitive, slide-based technique that incorporates the benefits of in situ polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) to quantify differential c-myc gene expression from liver tissue sections having either low or high levels of proliferating hepatocytes. To eliminate the need for isolating and quantifying mRNA, cells of interest were microdissected from frozen histological sections and their RNA directly subjected to RT-PCR amplification. These reactions were conducted in the presence of an internal RNA standard that was specifically designed to normalize differential RT and PCR efficiencies between samples. GENESCAN software analysis was used to determine the ratios of the RT-PCR products of the target gene to the RNA standard. These ratios were then normalized to the numbers of cells isolated, as quantified by image analysis, and comparative gene expression values were determined between sample groups. We conclude that this technology can be adapted to study any gene of interest in any type of frozen tissue or isolated cells. This methodology is particularly applicable to the molecular analysis of histopathologically distinct preneoplastic and neoplastic lesions identified on tissue sections.
Animals↗
Encapsulated genetically engineered fibroblasts: release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions.
Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable "minifactories" continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving "empty" alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs non-lesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased (P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.
Alginates↗
Evaluation of the predictive value of Her-2/neu gene expression on osteosarcoma therapy in laser-microdissected paraffin-embedded tissue.
Histologic response to chemotherapy is currently the strongest prognostic factor in high-grade osteosarcoma, but it can only be assessed after several weeks of therapy. Thus, detection of chemosensitivity at the time of diagnosis would be of great clinical importance. The expression of the proto-oncogene Her-2/neu has been shown to be of predictive value in breast cancer and has also been considered as prognostic marker for osteosarcomas, but reports of mainly immunohistochemical studies are controversial. Therefore, the aim of this study was to investigate Her-2/neu gene expression in laser-microdissected osteosarcoma cells. Laser microdissection enables the precise isolation of morphological defined cells from archival tissue specimens and is in combination with the highly sensitive real-time RT-PCR technique a valuable tool for cell-specific analysis of gene expression. Through optimization of current protocols, we could show that this technique can be successfully applied on formalin-fixed, paraffin-embedded and decalcified osteosarcoma tissue with high sensitivity and reproducibility. In all 17 osteosarcoma biopsies analyzed, we could detect Her-2/neu gene expression. Expression correlated significantly with the response to preoperative chemotherapy, which was assessed histologically according to the six-grade scale of Salzer-Kuntschik. Risk assessment on the basis of increased Her-2/neu gene expression matched the histologic findings in 16 out of 17 cases (94%). These data demonstrate the reliability of laser microdissection in the analysis of gene expression and suggest a possible role of Her-2/neu as prognostic marker for therapy outcome in osteosarcomas.
Antineoplastic Agents↗
Laser capture microdissection MALDI for direct analysis of archival tissue.
MALDI mass spectra were obtained from cancer cells isolated by laser capture microdissection (LCM) of archived tissue. Frozen human lung tissue from adenocarcenoma and squamous cell carcenoma cases were cut into 5 to 15 microm thick sections, stained with hematoxylin and dehydrated. Cancer cells were isolated by LCM, mixed with matrix solution, and deposited on a MALDI target for mass spectrometric analysis. For comparison with LCM isolated cells, tissue sections were placed directly on the MALDI target without microdissection. Tissue sections frozen in optimal cutting temperature (OCT) solution and cut into 8 microm thick sections gave the best performance with direct MALDI analysis. Between 15 and 20 peaks were observed in the mass region between 1,000 and 4,000 Da, and roughly half of these peaks were common to either squamous cells or adenocarcenoma. Additional peaks were observed in the non-LCM mass spectra and these may result from biomolecules in the healthy tissue. When compared to fresh tissue, both LCM and non-LCM archived tissue produced fewer peaks, possibly due to degradation of the biomolecules in the archived tissue.
Adenocarcinoma↗
Regional analysis of p53 mutations in rheumatoid arthritis synovium.
The p53 tumor suppressor protein plays a central role in cell cycle regulation, DNA repair, and apoptosis. Recent studies indicate that DNA damage and somatic mutations in the p53 gene can occur because of genotoxic stress in many tissues, including the skin, colon, and synovium. Although somatic mutations in the p53 gene have been demonstrated in rheumatoid arthritis (RA) synovial tissue and synoviocytes, no information is available on the location or extent of p53 mutations. Using microdissected RA synovial tissue sections, we observed abundant p53 transition mutations, which are characteristic DNA damage caused by oxidative stress. p53 mutations, as well as p53 mRNA expression, were located mainly in the synovial intimal lining rather than the sublining (P < 0.01). Clusters of p53 mutant subclones were observed in some microdissected regions, suggesting oligoclonal expansion. Because IL-6 gene expression is regulated by wild-type p53, IL-6 mRNA expression in microdissected tissues was quantified by using real-time PCR. The regions with high rates of p53 mutations contained significantly greater amounts of IL-6 mRNA compared with the low mutation samples (P < 0.02). The microdissection findings suggest that p53 mutations are induced in RA synovial tissues by inflammatory oxidative stress. This process, as in sun-exposed skin and inflamed colonic epithelium, provides some of the mutant clones with a selective growth advantage. A relatively low percentage of cells containing p53 mutations can potentially affect neighboring cells and enhance inflammation through the elaboration of proinflammatory cytokines.
Arthritis, Rheumatoid↗
Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.
Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.
Astrocytoma↗
Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.
The analysis of tissue-specific genetic alterations depends on the selective procurement of homogeneous cell populations. Microbeam microdissection of membrane-mounted native tissue (MOMeNT) permits the rapid, selective, and low-contamination procurement of tumor or other cells from histological sections by non-thermic non-contact laser microdissection. Tissue sections are mounted on a specifically designed ultrathin transparent supporter membrane. Tissue together with the membrane are then dissected with an ultraviolet (337-nm) pulsed laser microbeam coupled into a robot-stage microscope. The ultraviolet laser causes dissection by cold photolysis due to the high photon density of the microbeam rather than by local heating. The track of the laser microbeam can be preselected freely on a video screen, and the size and form of the dissectates can thus be adapted to the histological features of the section with a delineation accuracy in the micron range. Polymerase chain reaction amplification of DNA from the dissectates is not impaired, and tumor-specific loss of heterozygosity of the APC gene as well as homozygous deletion of the MTS1 gene are demonstrated in bladder carcinomas. Taken together, microbeam MOMeNT is a novel technique that utilizes membrane-based microdissection by an ultraviolet laser microbeam, thus providing a flexible, easy-to-use high-performance tool for the molecular pathologist.
Carcinoma, Transitional Cell↗
Leptin does not fully account for the satiety activity of adipose tissue-conditioned medium.
To determine whether leptin alone accounts for the satiety activity secreted by native adipose tissue, we prepared culture media conditioned by microdissected adipose tissue from overfed Long-Evans rats, fa/fa rats, or db/db mice (media A, B, and C, respectively). Medium A significantly suppressed food intake following intracerebroventricular delivery to Long-Evans rats (2-h chow intake = 68 +/- 5% of baseline, P < 0.001). Media B and C significantly suppressed food intake following intraperitoneal delivery to ob/ob mice (24-h chow intake = 56 +/- 7% of baseline for medium B, P = 0. 001; 4-day chow intake = 78 +/- 3% of baseline for medium C, P = 0. 004). Using a leptin receptor-based bioassay, we determined that the leptin concentration of medium C was 392 +/- 18 ng/ml. This concentration was 20-fold lower than the concentration of recombinant murine leptin required to produce a similar degree of feeding suppression following 5 days of administration to ob/ob mice. Neither medium conditioned by adipose tissue from ob/ob mice nor medium conditioned by adipose tissue from fa/fa rats and subsequently immunodepleted of leptin had significant satiety activity. We conclude that leptin is necessary but not sufficient to account for the satiety activity of native adipose tissue, perhaps due to the production by adipocytes of a cofactor that augments the ability of leptin to suppress feeding.
Adipose Tissue↗
Neuropeptide Y regulation of LHRH release in the median eminence: immunocytochemical and physiological evidence in hens.
It has been suggested that hypothalamic median eminence (ME) might be a control site for luteinizing hormone-releasing hormone (LHRH) release. Thus, stimulatory and/or inhibitory inputs acting at this site might be involved in regulating LHRH release from the ME and, therefore, luteinizing hormone (LH) release from the anterior pituitary. Since a role for neuropeptide Y (NPY) on LH release has been suggested, we have hypothesized that NPY might act in the ME to control preovulatory LHRH release in hens. To examine this possibility we have determined: (a) the immunocytochemical distribution of LHRH and NPY in the ME of the hen, (b) the basal and NPY-stimulated release of LHRH in vitro from the ME of hens undergoing a natural or a premature preovulatory surge of LH, and (c) the tissue content of LHRH and NPY in microdissected MEs, at various times before and during a natural or a premature preovulatory surge of LH. A potential role for NPY on LHRH release in the ME is suggested for the following reasons. (a) There are opportunities for synaptic interactions between NPY and LHRH-containing axons at this site. LHRH-containing cell bodies localized in the anterior hypothalamus/medial preoptic area project to the ME. NPY-containing perikarya, concentrated in the ventromedial aspect of the arcuate nucleus, might contact LHRH processes going to the ME and/or might themselves send axons to the ME, (b) Addition of NPY to the incubation media increases LHRH release from microdissected ME tissue of hens killed at the time of the natural preovulatory surge of LH, but not in hens killed 7 h before the occurrence of this surge. However, the stimulatory effect of NPY on LHRH release can be induced at this latter time when a premature LH surge is elicited. While the natural preovulatory surge of LH occurs 4 h before the second ovulation in a sequence (C2 ovulation), administration of progesterone (P4) 10-14 h before the expected natural C2 ovulation advances the natural LH surge by 7-8 h. Thus, NPY might act as a physiological stimulus of LHRH release at the ME during the preovulatory surge of LH. This is suggested since in vitro basal LHRH release from denervated ME tissue does not change before and during the natural or the premature LH surge. Therefore, preovulatory release of LHRH in vivo might be under a continuous drive from stimulatory inputs to the LHRH neuron and NPY might be one of these stimulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
In vitro culture of microdissected rat nasal airway tissues.
The surface epithelium lining the nasal airways is a potential target for inhaled contaminants such as ozone, endotoxin, formaldehyde, tobacco smoke, and organic dusts. The epithelial response to injury may depend on the toxicant, the type of epithelium, the severity of the injury, and the presence of inflammatory cells and their secreted products. To study mechanisms of toxicant-induced epithelial injury and repair, in the absence of cellular inflammation or other systemic effects, we have developed a culture system to maintain morphologically distinct nasal airway epithelium in vitro. Microdissected maxilloturbinates and proximal nasal septa of male F344/N rats were cultured at an air-liquid interface for up to 14 d in supplemented serum-free medium. Maxilloturbinates are lined by nonciliated cuboidal nasal transitional epithelium (NTE) with few or no mucous cells. The proximal nasal septum is lined by a mucociliary respiratory epithelium (RE) that normally contains numerous mucous cells. Preservation of the normal RE and NTE phenotype in culture was assessed by light and electron microscopy, and analysis of an airway mucin gene (rMuc-5AC) messenger RNA (mRNA). Both RE and NTE retained normal cell morphology for 14 d in culture (DIC). After 14 DIC there were 20% fewer RE cells in the septa (equal loss of ciliated and mucous cells) and 25% more NTE cells in the maxilloturbinates (increased number of basal cells). Compared with the RE, the NTE expressed consistently low levels of rMuc-5AC mRNA and had little to no histochemically detectable intraepithelial mucosubstances (IM) after 0, 3, 7, or 14 DIC. The amount of stored IM and the steady-state levels of rMuc-5AC mRNA in the RE decreased with time in culture. In summary, this culture system can maintain fully differentiated secretory and nonsecretory rat airway epithelia in vitro for up to 14 d. This study was an essential first step in developing a system to study the pathogenesis of toxicant-induced airway epithelial injury and mechanisms of cellular repair and adaptation in the absence of cellular inflammation and other systemic influences.
Animals↗
Multiple mechanisms generate HLA class I altered phenotypes in laryngeal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21.
Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.
Antigens, Neoplasm↗
Coordinated downregulation of the antigen presentation machinery and HLA class I/beta2-microglobulin complex is responsible for HLA-ABC loss in bladder cancer.
Downregulation of MHC class I expression is a widespread phenomenon used by tumor cells to escape antitumor T-cell-mediated immune responses. These alterations may play a role in the clinical course of the disease. The aim of our study was to investigate the molecular mechanism underlying the absence of HLA-class I molecule expression in bladder cancer cells. Microdissected tumor tissues were characterized by real-time quantitative PCR for the expression of HLA-ABC, beta2-microglobulin and the members of the antigen processing machinery (APM) of HLA class I molecules (LMP2, LMP7, TAP1, TAP2 and tapasin). Our results showed that irreversible HLA loss by mutations in the beta2-microglobulin gene was not the cause of low HLA class I expression in bladder cancers. In contrast, we observed a coordinated transcription downregulation of HLA-ABC and beta2-microglobulin and APM genes in microdissected tumor tissue derived from bladder carcinomas. This mechanism may represent a major factor for the downregulation of HLA class I expression and in the subsequent direct recognition of cancer cells by cytolytic T lymphocytes. Because this regulatory mechanism is frequently reversible by IFN-gamma treatment, we conclude that HLA class I expression should be a major consideration for immunotherapeutic purposes in patients with bladder cancer.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Transient coexpression of desmin and cytokeratins 8 and 18 in developing myocardial cells of some vertebrate species.
During myogenesis the intermediate-sized filament (IF) cytoskeleton is characterized by increasing proportions of desmin. While skeletal and smooth muscle formation occurs in free mesenchymal cells containing vimentin-type IFs, myocardial development starts from a polar epithelium containing cytokeratin IFs and desmosomes. Therefore, we have studied the formation of the epicardium and the myocardium in different vertebrate species, combining light and electron microscopic immunolocalization techniques with gel-electrophoretic analyses of cytoskeletal proteins of microdissected myocardial tissue at differing developmental stages. In this report, we describe results obtained from advanced stages of myocardial differentiation. In all species studied the myocardial cell possess IFs abundant in desmin, often together with smaller amounts of vimentin, and the mesothelial layer of the epicardium contains cytokeratin IFs. However, we have observed remarkable interspecies differences with respect to the occurrence of cytokeratins in embryonic myocardial cells. In fetal human myocardium, from week 10 of pregnancy on, but not in juvenile and adult myocardium, and in chicken myocardium of all stages examined (until several days after hatching) specific immunostaining was seen with certain broad-range cytokeratin antibodies as well as with antibodies specific for cytokeratins 18 (in both species) and 8 (showing significant reaction only in human). This cytokeratin immunoreaction, however, did not appear in IFs extending throughout the cytoplasm or at Z-lines, but was localized in punctate arrays representing aggregates of dense material. The aggregates were often enriched at, but not restricted to, the desmosomal plaques of the intercalated discs. These observations were supported by gel-electrophoretic demonstration of small but significant amounts of cytokeratins 18 (in both species) and 8 (detected only in human) in microdissected myocardial tissue. We also observed cytokeratins in smooth muscle cells of some cardiac blood vessels. In contrast, bovine myocardium of advanced fetal age as well as rat and mouse myocardium (from fetal day 12 on) were negative for cytokeratins with all methods, although epicardial cytokeratin IFs were demonstrable. These observations are discussed in relation to myocardial histogenesis and to general problems of cytokeratin gene expression control in epithelial and nonepithelial cells.
Animals↗
Multilineage progression of genetically unstable tumor subclones in cutaneous T-cell lymphoma.
Molecular analysis of solid malignant tumors has suggested multilineage progression of genetically unstable subclones during early stages of tumorigenesis as a common mechanism of tumor cell evolution. We have investigated whether multilineage progression is a feature of cutaneous T-cell lymphoma (CTCL). To identify individual tumor cell subclones, we determined the pattern of mutations within microsatellite DNA obtained from multiple histomorphologically confined tumor cell nests of mycosis fungoides (MF) and lymphomatoid papulosis (LyP) lesions. Tumor cells were isolated by laser microdissection, and allelotypes were determined at microsatellite markers D6S260, D9S162, D9S171, D10S215, TP53.PCR15, and D18S65. Nine cases of MF and one patient with anaplastic large cell lymphoma (ALCL) originating from LyP were analyzed at 277 different microdissected areas obtained from 31 individual lesions. Three specimens of cutaneous lichen planus microdissected at 26 areas served as the control tissue. Microsatellite instability in microdissected tissue [MSI(md-tissue)] was detected in tumor tissues of all CTCL patients. One hundred and fifty-seven of 469 analyzed polymerase chain reaction (PCR) amplifications contained mutated microsatellite alleles (34%). In lichen planus, MSI(md-tissue) was seen in only four of 76 PCR products (5%) (P < 0.0001). The distribution of allelotypes in tumor cells from different disease stages was consistent with multilineage progression in five MF cases, as well as in the LyP/ALCL patient. Our results suggest that CTCL may evolve by multilineage progression and that tumor subclones in MF can be detected in early disease stages by mutation analysis of microsatellite DNA obtained from multiple microdissected areas.
Alleles↗
The use of microsatellite instability in the distinction between synchronous endometrial and colonic adenocarcinomas.
The association of endometrial carcinoma with other gynecologic neoplasms, especially ovarian and fallopian tube carcinoma, has been well documented and is usually interpreted as a result of a field defect. Sporadic synchronous primary carcinomas occurring in the endometrium and colon are extremely rare, especially in the absence of the familial genetic abnormalities seen in hereditary nonpolyposis colorectal carcinoma (HNPCC) syndrome, and may present a diagnostic dilemma. Two cases of synchronous adenocarcinomas of the endometrium and colon were studied for genetic abnormalities and differences to test for the presence of two primary tumors. Primary tumors, metastases, and normal tissues were microdissected from formalin-fixed, paraffin-embedded tissues. PCR amplification was performed for microsatellite DNA markers on chromosome 17q and 11q13. The colonic tumors were moderately and poorly differentiated, invasive, nonmucinous adenocarcinomas, whereas one uterine tumor was endometrioid adenocarcinoma and the other was papillary serous carcinoma. Although microsatellite instability, as evidenced by changes in the lengths of the amplified PCR products, was detected at 17q and 11q13 loci in the uterine and colonic neoplasms, the patterns of instability differed between the two primary tumor sites. Moreover, the lymph node metastasis in one colonic tumor had genetic alterations that differed from that of the primary tumor. In both patients, the molecular studies suggested the presence of two synchronous primary tumors. Molecular techniques may assist in distinguishing two separate primaries by determining the contraction and expansion of microsatellite regions in DNA obtained by microdissection from the primary tumors and associated metastases.
Adenocarcinoma↗
Western blot analysis of a limited number of cells: a valuable adjunct to proteome analysis of paraffin wax-embedded, alcohol-fixed tissue after laser capture microdissection.
In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteome analysis largely depends on highly sensitive protein detection methods. In this study, a western blot protocol was developed and validated for the detection of beta-actin and the moderately expressed cell death protein caspase-3 in small numbers of cells. Initially, cultured human U937 monocytes and whole sections of paraffin wax-embedded, alcohol-fixed human tonsils were used to optimize protein electrophoresis and western blotting conditions. High-performance NuPAGE Bis-Tris gels in combination with high-quality transfer membranes, optimized antibody concentrations, and a sensitive chemiluminescent substrate provided a strong signal for beta-actin with approximately 500 U937 cells. In the same way, procaspase-3 could be identified with approximately 1000 cells. Similar results were obtained with germinal centre cells that were procured from paraffin wax-embedded, alcohol-fixed human tonsils by LCM. Treatment of U937 cells with etoposide rapidly induced cell death and allowed the detection of active caspase-3 with approximately 2500 cells (0.8 pg of protein). The findings of this study suggest that western blotting is a valuable adjunct to proteome analysis of LCM procured cells.
Actins↗
ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions.
BACKGROUND: Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, including malignant melanoma, prostate cancer cell lines, and prostate cancer. We aimed to clarify the expression patterns of ALCAM in normal and malignant prostate tissue. METHODS: RNA from 54 matched pairs of microdissected prostate tissue (tumor and normal) was hybridized to a custom built 4K GeneChip. Frozen section immunohistochemistry was used to confirm the ALCAM expression. RESULTS: Chip-based transcript analysis revealed an up-regulation of ALCAM in prostate cancer in 22% of cases. Immunohistochemistry showed a focally raised ALCAM expression in 81% and a decreased expression in 19% of cases. These expression patterns correlated significantly with Gleason tumor grade: ALCAM up-regulation was found in most low-grade tumors (Gleason grade 1-3), whereas down-regulation occurred preferentially in high-grade tumors (Gleason grade 4 and 5), although up-regulation of ALCAM expression was preserved in two Gleason grade 5 tumors. CONCLUSION: ALCAM expression is commonly disturbed in prostate cancer, which might indicate a role of ALCAM in the progression of this disease.
Activated-Leukocyte Cell Adhesion Molecule↗