p53 expression in oat and non-oat small cell lung carcinomas: correlations with proliferating cell nuclear antigen.

AIMS--To investigate the immunohistochemical expression of p53 protein in small cell lung carcinoma (SCLC), comparing it with that in non-small cell carcinoma (NSCLC); and to evaluate the correlation between p53 and proliferating cell nuclear antigen (PCNA) expression as well as between p53 and PCNA expression and survival. METHODS--Paraffin wax embedded tissues from 61 cases of primary lung carcinoma were stained for p53 protein and PCNA using the monoclonal antibodies 1801 and PC-10, respectively, in a standard avidin-biotin immunoperoxidase method. RESULTS--Of the 61 lung carcinomas 35 (57%) were positive for p53 (range 1% to 90%). Ninety percent of the non-oat SCLC contained positive cells and the labelling index (LI) was significantly higher than that of the oat SCLC (p < 0.001). SLCC also displayed a higher p53 LI than NSCLC (p < 0.01); no difference was found between squamous carcinoma, adenocarcinoma, and oat cell carcinoma. A p53 LI of greater than 1% tended to be associated with nodal metastases (p < 0.05), and p53 expression in node positive tumours as well as in oat cell carcinomas was indicative of poorer survival (p < 0.01 and p < 0.1, respectively). A p53 Li of greater than 60% was a negative prognostic factor in non-oat SCLC (p < 0.05). PCNA LI was highest in non-oat SCLC and lowest in NSCLC; oat cell carcinomas had a mean LI intermediate between NSCLC and non-oat SCLC (NSCLC v oat cell carcinoma p < 0.05 and oat cell v non-oat cell carcinomas p < 0.01). A PCNA LI was not correlated with nodal metastases or survival, but there was a significant positive correlation between PCNA LI and p53 LI (rs = 0.484, p < 0.001). CONCLUSIONS--p53 and PCNA expression differ substantially among the various types of lung carcinomas. Substantial differences were also found between oat and non-oat types of SCLC, indicating that SCLC is heterogeneous as far as proliferation rate and altered p53 expression is concerned. p53 seems to be of some prognostic value. The relation between PCNA and p53 expression indicates that the PCNA gene is slightly upregulated by p53.     

Exocytosis of neurosecretory granules in small cell undifferentiated carcinoma

The tumor cells in the pleural effusions of a case of small cell undifferentiated carcinoma have been studied by light microscopy and by thin section and freeze-fracture electron microscopy. Evidence of exocytosis of neurosecretory granules from the tumor cells is presented. The tumor cells form clumps, with the cells joined by focal tight junctions and small desmosomes. These findings are discussed with reference to the neuroendocrine origin of these tumors and the role of the junctional complexes in the formation of tumor cell aggregates.     

A second sensory--motor--interneuron with neurosecretory granules in Hydra.

Summary Using serial-sectioning techniques for conventional transmission and high-voltage electron microscopy, we characterized the ultrastructural features and synaptic contacts of the sensory cell in tentacles ofHydra. The sensory cell has an apical specialization characterized by a recessed cilium surrounded by three rodlike stereocilia. This ciliary—stereociliary complex constitutes the receptive or dendritic pole of the sensory cell. The dense filamentous cores of the stereocilia project proximally into a narrow circumciliary cytoplasmic region connected by septate junctions to marginal processes of an enveloping epitheliomuscular cell. The central cilium has a characteristic marginal flare midway along its length and a dense filamentous substructure at its base. Pairs of branched, striated rootlets extend from the axial centriole into a mitochondria-rich region of the cell. Pigment-like granules are present in the cytoplasm around the circumciliary space. The perikaryon is characterized by an elongate nucleus surrounded by a narrow rim of cytoplasm containing prominent Golgi complexes, assorted vacuoles and dense-cored vesicles, free ribosomes, short segments of rough endoplasmic reticulum, microtubules, glycogen particles, and lipid droplets.Generally, one or two thin, naked axons extend laterally from the perikaryon into the nerve net region above the myonemes of the large epitheliomuscular cells. Within the axons are found occasional aggregates of dense-cored vesicles anden passant synapses characterized by the presence of clear or dense-cored vesicles in contact with paramembranous densities and associated intracleft cross filaments. Using these ultrastructural criteria, we demonstrated for the first time that the granule-containing sensory cells have synaptic contacts with other neurons, nematocytes, and epitheliomuscular cells; hence, we considered these cells to be sensory–motor–interneurons with neurosecretory granules. We hypothesize that this unique, apparently multifunctional neuron may be a modern representative of a primitive stem cell that gave rise evolutionarily to the sensory cells, motor neurons, interneurons, and neurosecretory cells of higher animals.     

EB病毒膜蛋白在CHO细胞中的表达

目的研制ＥＢ（Ｅｐｓｔｅｉｎ－Ｂａｒ）病毒基因工程疫苗。方法构建了含有Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）膜蛋白（ＭＡ）基因的重组表达质粒ｐＣＭＶ／ＭＡ，转染ＣＨＯ细胞，研究表达产物的生物学性状，及培养液中不同血清含量、冻存、Ｇ４１８对ＣＨＯ细胞分泌ＭＡ的影响。结果获得两个稳定表达ＭＡ的细胞株。免疫印迹检测表达产物的分子量约为３４０ｋＤ和２２０ｋＤ，间接免疫荧光和免疫斑点确定表达产物能与抗ＭＡ的单克隆抗体特异性结合，用薄层扫描和Ｌｏｗｒｙ法计算ＭＡ的表达量为每天１９μｇ／ｍｌ。经纯化的ＭＡ免疫小鼠，在血清中检测到抗ＭＡ的特异性抗体。结论为开发有效的表达系统用于ＥＢ病毒基因工程疫苗的生产提供有利的实验基础。     

Expression of MAGE tumor-associated antigen in thyroid carcinomas

The 12 members of the MAGE gene family encode tumor-specific antigens that are recognized by autologous cytotoxic T lymphocytes. The MAGE genes are expressed not only in melanoma but in other malignant tumors as well. There is, however, little information on their expression in thyroid carcinomas. We studied the expression of the MAGE-3 antigen in human thyroid carcinomas to explore the possibility of specific immunotherapy using MAGE peptides. Tumor tissue samples of thyroid carcinomas were obtained from 60 patients. Standard pathohistologic analysis followed by immunohistochemistry analysis of MAGE-3 expression was performed in all patients. The overall expression rate of MAGE-3 antigen in thyroid carcinomas was 65%. According to histological types of thyroid carcinomas, expression rate of MAGE-3 antigen was as follows: 0% in anaplastic, 20% in medullary, 29% in follicular, and 80% in papillary thyroid carcinomas (p<0.01). On the other hand, significantly higher expression of MAGE-3 antigen was observed in classical subtypes of papillary thyroid carcinomas and in small papillary tumors sized to 1 cm in diameter. These findings demonstrated that MAGE-3 antigen expression seems to be particularly high in the small, typical papillary carcinomas, thus suggesting that MAGE-3 gene abnormality is an early step in thyroid cancer progression.     

Expression of the pS2 peptide in primary breast carcinomas: Comparison of membrane and cytoplasmic staining patterns

The pS2 protein is oestrogen-regulated in breast cancer cell lines. Previous studies have shown a relationship to oestrogen receptor in primary breast carcinomas. This study examined 178 breast carcinomas for pS2 using immunohistochemistry. A high frequency (77 per cent) of positive tumours was found, using a 10 per cent cut-off point to define a positive tumour. There was no relationship with menopausal status or node status, a significant association with differentiation, a weak association with oestrogen receptor, and no association with progesterone receptor or overall survival. Two patterns of cellular localization were observed: cytoplasmic and membrane. The former showed a stronger relationship with oestrogen receptor status, although there were oestrogen receptornegative tumours with marked pS2 staining. Membrane staining showed a stronger relationship with differentiation, with a staining pattern similar to that observed for milk fat globule membrane. The staining patterns observed may support a role for pS2 in a secretory mechanism. However, the expression and function of pS2 in breast carcinomas emain complex, and are not simply related to oestrogen regulation.     

Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation.

Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increased following stimulation of the cells, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils.     

Expression of prostate-specific membrane antigen in renal cortical tumors.

Prostate-specific membrane antigen is a type II membrane glycoprotein that is expressed in benign and neoplastic prostatic tissue and has been recently shown to be also expressed in the neovasculature of various solid malignant tumors including renal cell carcinoma. Renal cell carcinoma is a heterogeneous group of tumors with distinct morphologic and genetic characteristics and clinical behaviors. We performed immunohistochemical studies on formalin-fixed, paraffin-embedded archival material from 75 nephrectomies, using antibodies 13D6 against prostate-specific membrane antigen and CD31 against endothelial cells. The study included 30 clear cell renal cell carcinomas, and 15 of each of papillary and chromophobe renal cell carcinoma and oncocytoma. The extent and intensity of staining were assessed semiquantitatively. In all cases, immunoreactivity was detected only in the tumor-associated neovasculature and not in tumor cells. Clear cell renal cell carcinoma showed the most diffuse staining pattern, where 24/30 cases or 80% had >50% reactive vessels, followed by chromophobe renal cell carcinoma (9/15; 60%) and oncocytoma (5/15, 33%). No diffuse staining was detected in any of the papillary renal cell carcinomas and only focal staining was detected in 11 cases (11/15; 73%). Staining intensity was the strongest in clear cell renal cell carcinoma (25/30; 83%) followed by chromophobe renal cell carcinoma (9/15; 60%), oncocytoma (8/15, 53%) and papillary renal cell carcinoma (5/15; 33%). In summary, prostate-specific membrane antigen is expressed in tumor-associated neovasculature of the majority of renal cortical tumors and is most diffusely and intensely expressed in clear cell renal cell carcinoma and least in papillary renal cell carcinoma. The differences in the expression of prostate-specific membrane antigen in renal cell carcinoma subtypes provide further evidence of the biological diversity of these tumors, and diagnostic and therapeutic applications of such expression can be expanded to include subtypes of renal cell carcinoma.     

转染CRKL蛋白对肺癌细胞系增殖的研究

目的研究CRKL蛋白对肺癌细胞系增殖的影响及其可能机制,探讨CRKL基因在肺癌发病中的作用。方法在转染CRKL的HBE和H1299两种肺癌细胞系中,采用Western blot,流式细胞技术(FCM),集落形成实验和MTT方法检测转染CRKL后,对肺癌细胞增殖的影响。结果转染CRKL基因后,可明显上调Cyclin D1和Cyclin B蛋白在HBE和H1299两种肺癌细胞表达,增加Rb的磷酸化,促进肺癌细胞的增殖、克隆形成能力。结论 CRKL通过调节细胞周期促进肺癌细胞的增殖能力,CRKL可作为非小细胞肺癌的标记物,并可能成为治疗该肿瘤的新靶点。     

Expression of cyclo-oxygenase-2 is correlated with high intratumoral microvessel density and low apoptotic index in human esophageal squamous cell carcinomas

Cyclo-oxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostanoids. COX-2 expression has been found in many malignancies. This study analyzed the correlation between COX-2 expression and angiogenesis or apoptosis in human esophageal carcinomas. The study examined the expression of COX-2 in six esophageal carcinoma cell lines and in 100 esophageal squamous cell carcinomas, comparing intratumoral microvessel density (IMVD) and apoptotic index (AI) by immunohistochemistry and TUNEL methods. COX-2 was variably expressed in all the cell lines examined. COX-2 immunoreactivity was observed mainly in the cytoplasm of carcinoma cells. Significantly higher mean IMVD and lower AI were noted in the 51 strong COX-2 expressing cases than in the 49 weak cases. IMVD and AI were negatively correlated. COX-2 expression was higher in the tumors with lymphatic invasion than in the others. These data indicate that COX-2 expression is associated with increased intratumoral microvessels and suppression of tumor cell apoptosis. Thus COX-2 might play an important role in the angiogenesis and regulation of apoptosis in esophageal squamous cell carcinomas.     

Expression of tumor-associated membrane antigen, RCAS1, in human colorectal carcinomas and possible role in apoptosis of tumor-infiltrating lymphocytes.

RCAS1, a novel tumor-associated antigen, is expressed in advanced human neoplasias including uterine and ovarian carcinomas. RCAS1 protein was indicated to induce cell cycle arrest and apoptosis of cultured human lymphoid and myeloid cell lines and normal lymphocytes. In the present study, we investigated the expression and prognostic value of RCAS1 in 58 patients with colorectal carcinomas. RCAS1 protein was detected by immunoperoxidase staining using a mouse monoclonal anti-RCAS1 antibody (22-1-1 antibody). Immunohistochemical examination showed expression of RCAS1 in 75% of colorectal carcinomas with lymph node metastases (n = 24), whereas it was present in only 41% of tumors without metastases (n = 34, P <.05). Patients with RCAS1-positive tumors showed a significantly poorer prognosis than those negative for RCAS1 (P <.05). Multivariate analysis using the Cox regression model indicated that RCAS1 positivity was an independent negative predictor for survival (P =.0300; risk ratio, 0.496). In addition, apoptotic cells of tumor-infiltrating lymphocytes were examined using nonradioactive in situ nick translation in paraffin-embedded sections. The proportion of apoptotic tumor-infiltrating lymphocytes was significantly higher in RCAS1-positive colorectal carcinomas (11.2 +/- 1.0) than in RCAS1-negative tumors (7.9 +/- 1.0, P <.05). Our results suggest that overexpression of RCAS1 may negatively affect the prognosis of human colorectal carcinomas and that RCAS1 may play a role in tumor immune privilege in vivo.     

Expression of Tac antigen in B cell lymphomas.

In a series of 55 cases of B cell derived non-Hodgkin's lymphoma the reactivity of two distinct anti-Tac monoclonal antibodies was examined using a sensitive immunoperoxidase technique on cryostat sections. Eighteen out of the thirty-five cases of B cell lymphomas of low or intermediate grade of malignancy were found to be reactive while six out of 20 cases of high-grade malignancy lymphomas showed a positive immunostaining. No correlation was found between anti-Tac reactivity and surface immunoglobulin phenotype, T65 antigen, or calla expression. These findings showed that IL2 receptor expression is not restricted to activated T cells, and raise the question of the possible role of IL2 in the regulation of malignant B cell clone expansion.     

Two cases of acute lymphoblastic leukemia with cytoplasmic granules

The presence of cytoplasmic granules in blastoid cells of patients with acute leukemia is generally accepted as a useful morphological marker for differentiation of myeloid leukemia from lymphoblastic leukemia. We diagnosed two cases of acute lymphoblastic leukemia(ALL) with cytoplasmic granulation. Surface marker analysis of leukemic cells revealed they were positive for CD10, 19, 20, 33, 34 and HLA-DR. Immunoglobulin gene rearrangement was detected by means of Southern hybridization with an Ig-JH probe for both patients. On the basis of these findings, the patients were diagnosed as having B-precursor ALL. Electron microscopic observation showed no myeloperoxidase activity, so that the granules were considered to be related to autophagolysosomes. This experience demonstrates that the recognition of the presence of granular ALL is necessary for making an accurate differential diagnosis of acute leukemias.     

Perinuclear and cytoplasmic distribution of desmoglein in esophageal squamous cell carcinomas

The desmosomal glycoproteins desmoglein (Dsg) and desmocollin (Dsc) are members of the cadherin family of cell adhesion molecules. They play an important role in epithelial adhesion. To observe the distribution pattern of Dsg in esophageal squamous cell carcinomas (SCC), immunohistochemical and immunoelectron microscopic analyses were performed. Immunohistochemically, normal esophageal squamous cells strongly expressed Dsg at the cell-cell boundaries, while moderately differentiated esophageal SCC cells showed a perinuclear distribution in addition to the cell boundary staining. At the ultrastructural level, the reaction product was concentrated at the desmosomes in the cell membrane region of normal epithelial cells, but was reduced at the membrane and found throughout the cytoplasm as well as in the surrounding outer nuclear envelope in SCC cells. These results demonstrate an aberrant distribution of Dsg in SCC cells. This may have important consequences for invasion and metastasis, as it may indicate loosened intercellular adhesion.     

Expression of Epstein-Barr-virus-associated membrane antigen in Raji cells superinfected with two different virus strains.

Papain treatment of Epstein-Barr virus (EBV; (P3HR-1 strain)-superinfected Raji cells removed the initially adsorbed membrane antigen (MA) positive material from the cell membranes. MA positive cells appeared again after 20 hr in culture, reaching a maximum level at about 30 hr. Puromycin, cycloheximide, and actinomycin D prevented the appearance of MA, whereas cytosine arabinoside had no effect. These results suggest that MA is synthesized de novo by P3HR-1 virus-infected cells and can be detected after 20 hr. The effects of the different metabolic inhibitors are in line with the concept that MA synthesis is an early function of the viral genome. Parallel EA induction tests showed that papain treatment had no effect on the frequency of EA positive cells compared to buffer-treated cells.A comparison between P3HR-1 and B95-8 virus strains with equal EBNA-inducing capacity showed that B95-8 virus was deficient with regard to its ability to induce MA in Raji cells, as judged by direct immunofluorescence.     

Ber EP4 and epithelial membrane antigen aid distinction of basal cell, squamous cell and basosquamous carcinomas of the skin

AIMS: Seventy-five skin tumours were studied to investigate the value of immunohistochemistry in differentiating basal cell, squamous cell and basosquamous carcinomas of the skin. METHODS AND RESULTS: Archived paraffin-embedded tissue samples of basal cell carcinomas (n = 39), squamous cell carcinomas (n = 23) and basosquamous carcinomas (n = 13) were stained immunohistochemically using a panel of antibodies. All of the basal cell carcinomas stained positively for Ber EP4, in contrast to the group of squamous cell carcinomas, that showed no staining. Basosquamous carcinomas all showed at least some areas of Ber EP4 positivity. None of the basal cell carcinomas, but most of the squamous cell carcinomas (22 of 23) expressed epithelial membrane antigen (EMA). Only one of the basosquamous carcinomas expressed EMA positivity focally. CAM 5.2, carcinoembryonic antigen (CEA) and 34betaE12 antibodies lacked specificity in relation to the different tumour types. CONCLUSION: Distinction of basal and squamous cell carcinomas of the skin can be readily achieved with routine immunohistochemistry using Ber EP4 and EMA. Identification of basosquamous carcinoma is also facilitated with this method.     

Chloride permeation across the Deiters' neuron plasma membrane: activation by GABA on the membrane cytoplasmic side.

Single plasma membranes were microdissected from Deiters' neurons freshly obtained from the lateral vestibular nucleus of the rabbit and their chloride permeability was studied in a microchamber system. The basal in-->out 36Cl- permeation initially found was brought to zero by Zn2+, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and iodide. GABA on the membrane cytoplasmic side resulted in a measurable in-->out 36Cl- passage, which was blocked by the GABA(A) antagonists bicuculline and picrotoxin. This effect peaked at 1 microM GABA on the inner side of the membrane. At higher GABA concentrations, a strong desensitization of the effect was found. Stimulation of Cl- permeability by GABA on the extracellular side of the membrane peaked at much higher GABA concentrations, 10-100 microM. This excludes an effect due to passage of the neurotransmitter from the inner to the outer compartment in our microchamber device. Moreover, this possibility is also dismissed by the fact that 1 microM GABA on the membrane outside did not evoke any 36Cl- in-->out permeation. In addition, pentobarbitone by itself could also stimulate 36Cl- in-->out permeation when added on the cytoplasmic side of Deiters' membrane. On these bases and in agreement with our previous reports, we propose that structures behaving pharmacologically as GABA(A) receptors respond to low levels of GABA on the cytoplasmic side of these neurons' membranes. We suggest that these structures are devices that, at the expense of ATP consumed in their phosphorylation, extrude Cl- after postsynaptic GABA uptake into the Deiters' neuron.     

Expression of Aleutian mink disease antigen in cell culture.

Infection of CRFK feline kidney cells with Aleutian disease vurus leads to production of virus-induced antigen(s) in the nucleus which could be demonstrated by the fluorescent-antibody technique. The number of fluorescent nuclei was lineraly dependent on the dilution of the inoculum, but rarely exceeded 20% of the cells. Aleutian disease nuclear antigen was only transiently detectable. The virus-induced antigen was detected after infection of cells of several divergent species; however, the CRFK line of feline kidney cells was the most susceptible. Inhibitor studies indicated that deoxyribonucleic acid synthesis, ribonucleic acid synthesis, and protein synthesis were required for viral antigen production. Cell growth was also a requirement for synthesis of viral antigen, An in situ radioimmune assay was used to measure binding of 125I-labeled mink anti-Aleutian disease virus to infected cells and competition with unlabeled sera. The system is suitable for quantitation of infectivity.