The nm23 gene, which encodes nucleoside diphosphate (NDP) kinase, is proposed as a metastatic suppressor gene. Loss of heterozygosity (LOH), and the expression of the nm23 gene were examined on matched sets of primary tumors and lymph node as well as distant metastases from colorectal carcinomas. Three (15%) of the 20 informative specimens examined showed LOH for the nm23 locus. nm23 was expressed in all the carcinomas as well as in nonneoplastic colonic mucosa at the mRNA and protein levels. Most of the carcinomas expressed the nm23 transcript at higher levels than the corresponding nonneoplastic colonic mucosa. By Western blotting, the level of nm23 protein expression in the tumors showed an inverse correlation with the tumor stage. Furthermore, distant metastatic tumors in the liver and lung showed reduced nm23 immunoreactivity in comparison with their primary tumor, although nm23 immunoreactivity was the same in the primary tumors and in local lymph node metastases. These results suggest that decreased nm23 expression may be associated with distant metastatic spread.
Using monoclonal antibodies against proliferating cell nuclear antigen or PCNA (PC10) and the Ki-67 antigen (MIB1), an immunohistological and morphometric study was performed on routinely processed splenic tissue from ten patients with primary (idiopathic) osteomyelofibrosis (OMF). To determine the proliferation capacity of erythroid precursors and the endoreduplicative activity of megakaryocytes, corresponding antibodies (Ret40f and CD61) were applied in combination with the cell-cycle markers (sequential double-immunostaining). Morphometric analysis revealed no significant differences in PCNA or Ki-67 reactivity in either cell lineages. In comparison with previous studies on normal bone marrow, in splenic tissue showing myeloid metaplasia, the numbers of PCNA-labelled proerythroblasts, erythroblasts and megakaryocytes were conspicuously increased. Considering the ineffective erythropoiesis in OMF, there seemed to be a disproportional enhancement in PCNA and Ki-67 immunostaining of the red cell lineage. Similarly, the small size of megakaryocytes in advanced, OMF-associated myeloid metaplasia was in keeping with an impairment of endoreduplicative activity. In addition to various other contributory factors, anaemia in OMF may be partially caused by secondary folate (haematinic) deficiency. From experimental studies this defect is known to cause an abnormal arrest in the S-phase of the cell-cycle, comparable to that characterising pernicious anaemia. As a sequel of this pathomechanism, an undue overexpression of PCNA and Ki-67 has to be assumed, that is not necessarily associated with DNA synthesis or cell cycling.
The distribution of proinsulin and insulin immunoreactivity was studied in 76 human insulinomas and in normal pancreas. One trabecular and two solid insulinomas showed the staining pattern of normal beta cells. A "near normal" staining pattern (perinuclear proinsulin and diffuse or polarized insulin staining) existed in 10 of 27 trabecular and 11 of 44 solid insulinomas. An "intermediate" staining pattern (intense perinuclear as well as weaker diffuse proinsulin staining with diffuse or polarized insulin staining) was observed in 10 of 27 trabecular and 20 of 44 solid insulinomas. Different "abnormal" staining patterns were found in 6 of 27 trabecular and 6 of 44 solid insulinomas. Of the 5 glandular insulinomas, 4 exhibited a "near normal" and one an "abnormal" staining pattern. No correlation was found between any particular staining pattern and the multihormonality or malignancy of the insulinomas. The diffuse labeling for proinsulin in about 50% of the insulinomas is suggestive of abnormal prohormone processing.
Using a panel of antibodies (Abs) and a lectin, normal human adult pituicytes were studied in neurohypophyses obtained from 29 patients at antopsy. The pituicytes reacted frequently with Abs against major histocompatibility complex (MHC) class II antigens (Ags), macrophage markers (KP.1, PG.M1, LN-5), an anti-vimentin Ab and a biotinylated lectinRicinus communis agglutinin (RCA-1). The number of pituicytes immunostained for these reagents varied, with the notable exception of vimentin. MHC class II Abs (LN-3, CR3.43)-positive pituicytes were numerous in approximately half. Microscopically, MHC class II Ag was found in pituicytes of various shapes, and were identified in macrophage-typed pituicytes by electron microscopic immunohistochemistry. Glial fibrillary acidic protein was found in only a small number of pituicytes and was absent in cells labeled with MHC class II Abs or macrophage markers. The results indicate that the immunophenotype of human pituicytes is distinct from other glial cells of the central nervous system, with a considerable number of cells expressing MHC class II Ags and macrophage markers.
The migration and maturation of Langerhans cells (LCs) in rat tracheal squamous metaplasia due to vitamin A deficiency were investigated immunohistochemically and electron microscopically. In the early stage of metaplasia, i.e. basal cell hyperplasia, no LCs with Birbeck granules (BGs) could be found, but there were desmosome-free cells which had the morphological charcteristics of immature LCs. They were clearly different from inflammatory cells such as macrophages and lymphocytes, and were, therefore, considered to be precursors of LCs. In the stage of stratification, small numbers of Ia- and protein kinase C type II (PKCII)-positive cells were recognized. Ultrastructually they were immature LCs with ovoid nuclei, many free ribosomes and few dendrites. The cytoplasm was dark and a few BGs and atypical granules (AGs) could be seen in the Golgi area. In the early stage of cornification, LCs with partially intended nuclei, prominent nucleoli and well-developed Golgi complexes were found. There were many BGs and AGs and structures transitional between them in the Golgi areas. In epithelium showing mature squamous metaplasia, many Ia- and PKCII-positive dendritic cells could be seen. Most of these were typical mature LCs with lobulated nuclei, clear cytoplasm and prominent dendritic processes. The number of BGs and AGs was fewer than in the LCs found in the early stage of cornification, and these granules were distributed throughout the cytoplasm. In the final stage, where the basal cells had differentiated into a flatter epithelium, few LCs could be seen. These findings suggest that the precursors of LCs without BGs migrate into metaplastic squamous epithelium and mature into LCs forming BGs after exposure to the microenvironment of the squamous epithelium.
The involvement of fat-storing cells (FSC) in hepatic schistosomal granuloma was investigated in mice infected with Schistosoma mansoni cercariae. After infection, 24 animals were treated with s.c. injections of vitamin A in a total dose of 210,000 IU given twice a week for 3 weeks. Two other groups of 24 animals each were: a) non-infected vitamin A-treated animals and (b) untreated infected controls. Animals from all groups were killed at weekly intervals from the 5th through the 10th week following infection. Pieces of liver were examined by light microscopy and transmission electron microscopy. In all vitamin A-treated animals, FSC disclosed prominent cytoplasmic fat droplets, which permitted their prompt identification in the light and in the electron microscope. They were found in large numbers as a constituent of periovular granulomas. In infected controls, FSC were not identified in granulomas, possibly because lipid droplets disappeared during differentiation to the fibroblastic phenotype. FSC also appeared within areas of septal fibrosis. These data suggest that FSC play an important part in focal portohepatic fibrosis during granuloma formation around S. mansoni eggs in the liver of mice.
Cells from the cysts of patients with autosomal dominant polycystic kidney disease (PKD) were grown in vitro under standard conditions without the aid of collagen-pretreated surfaces, and both the synthesis and composition of the extracellular matrix were investigated. At confluence, PKD cells presented the typical features of epithelial cells, but showed a different collagen composition from fibroblasts. Compared with normal tubular epithelia (NTE), PKD monolayers produced an excess of extracellular matrix, which accounted for 30% of the total incorporation of [3H] proline, although this value was considerably lower (by a factor of 10) in the case of NTE. Immunohistochemical and electrophoretic techniques revealed a complex collagen composition in the extracellular matrix which included [alpha (III)]3 and collagen IV. However, part of the collagen components remained unidentified in spite of the fact that they exhibited a typical M(r) of alpha 1(I) and alpha 2(I) in the presence of urea. Immunoprecipitation with monospecific antibodies and Northern blotting with specific probes failed to recognize alpha 1(I) and alpha 2(I), but demonstrated their presence in fibroblasts. Purification and cyanogen bromide digestion demonstrated a strong interhomology in fingerprint peptide composition among the uncharacterized collagens synthesized by PKD cells, thus suggesting a common identity. These observations document a markedly augmented production of extracellular matrix by PKD cultured cells in vitro, and show the presence of collagens which do not share homologies with the major collagen molecules. A better characterization of extracellular matrix composition is central to any comprehension of the cytogenetic mechanisms in vivo.
Smooth muscle differentiation has been analysed in human myometrium and leiomyoma by Western blotting with antibodies to smooth muscle specific proteins. No differences in the expression of h-caldesmon, metavinculin, desmin, α-smooth muscle actin and calponin were observed. The technique of two-dimensional gel electrophoresis was used, therefore, to further analyse differences between normal smooth muscle cells and their neoplastic counterparts. By comparing the protein patterns of normal myometrium and leiomyoma, it was possible to identify a protein with a molecular weight of approximately 27 kD that is selectively expressed in normal uterine smooth muscle cells. This protein proved to be a low molecular weight variant of calponin, a smooth muscle specific protein of as yet unknown function. Its immediate downregulation in tissue culture of normal myometrium points to a possible role in the process of dedifferentiation.
Amplification of genomic DNA encoding oncogenes such as HER-2 (syn. c-erbB2/c-neu) may be substantially involved in the initiation and progression of breast cancer. In order to refine and facilitate the quantitative analysis of HER-2 amplification in breast cancer, differential polymerase chain reaction (PCR) was performed on DNA derived from single cryosections of tumor tissue. This technique is based on the simultaneous amplification of a potentially amplified oncogene (HER-2) and a reference gene (IFN-gamma). Differential PCR yielded reproducible results that were in agreement with gene copy quantification using the dot blot technique. Thus we suggest differential PCR to be a reliable and rapid method for determining relative gene dosage in a minute amount of tumour tissue.
Morphologically atypical cells were first detected in the adjacent connective tissue 98 days after implanting a paraffin pill containing 2 mg of 7,12-dimethylbenz[a]anthracene (DMBA) into the subcutaneous tissues of rats. These cells subsequently formed groups and finally produced gross malignant fibrous histiocytomas (MFH). Early atypical cells were located between proliferating fibroblasts and histiocytes in the center of a fibrous capsule surrounding the DMBA pill. They exhibited a smooth cell surface, dilated rough endoplasmic reticulum, multiple Golgi complexes, and were often associated with newly formed collagen. These cells incorporated [3H]thymidine and [3H]proline intensively, and showed weak acid phosphatase activity but no features diagnostic of macrophages (microvilli, numerous lysosomes, high acid phosphatase and non-specific esterase activities, antigens recognized by monoclonal antibodies ED1 and OX-42 and vital staining with trypan blue). There was no evidence that atypical cells differentiated into muscle cells (no expression of desmin or the α-sarcomeric form of actin) or Schwann cells (no expression of S-100 protein). No point mutation in theneu gene at nucleotide 2007, specific for N-ethyl-N-nitrosourea-and DMBA-induced malignant rat schwannomas, was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analyses. These results support the view that malignant fibrous histiocytoma is derived from immature fibroblasts exhibiting pronounced phenotypic diversity during the later stages of carcinogenesis.
Myocardial peroxisomes were investigated in normal and diabetic rats. Catalase and acyl-CoA oxidase activities were increased in the diabetic rat heart and immunoblot analysis showed that both enzyme proteins were markedly enhanced in diabetic heart homogenates. After immunoenzyme staining, catalase and acyl-CoA oxidase were localized in fine granules in the myocardium, which were increased in number in diabetic rats. The numerical density of the granules stained for catalase was increased 1.7 times and that for acyl-CoA oxidase 1.8 times, compared with controls. Protein A-gold labeling for catalase and acyl-CoA oxidase was present in myocardial peroxisomes. The labeling density for both enzymes was increased in diabetic rats by 1.6 times for catalase and 1.5 times for acyl-CoA oxidase, compared with controls. The results indicate that myocardial peroxisomes are increased in the diabetic rat and that this proliferation is accompanied by an increase in catalase and acyl-CoA oxidase activities.
A new human epithelioid sarcoma cell line (ES020488) was established from a cutaneous metastasis in 26-year-old man, and was morphologically characterized in vitro and in vivo by comparison with the original tumor. The ES020488 cells showed a male karyotype ranging from 39 to 83 chromosomes, with various abnormalities but no specific pattern. The cells were round, polygonal or spindle-shaped with abundant cytoplasm and round nuclei containing prominent nucleoli; they proliferated in a sheet-like pattern. Tumors transplanted into nude mice revealed essentially the same features as the original tumor. Both in vitro and in vivo, the cells immunohistochemically expressed vimentin, cytokeratin, and EMA, but not desmin and S-100 protein. Ultrastructural study revealed irregular or round nuclei containing abundant euchromatin and prominent nucleoli, many intermediate filaments running irregularly or around the nucleus, and a number of filopodia-like processes. ES020488 cells were thus proven to retain and exhibit the unique morphological characteristics of an epithelioid sarcoma both in vitro and in vivo. These cells are possibly derived from synovioblastic mesenchyme.
Sequential changes in the expression of two glucose transporter isoforms (GLUT1, GLUT2), and in the activities of hexokinase, pyruvate kinase and malic enzyme during the development of rat renal basophilic cell tumors were studied using histochemical techniques. Early basophilic cell tubules are similar to proximal convoluted tubules (PCT) in their overall histochemical pattern, particularly in the expression of glucose transporters, suggesting that basophilic cell tubules and tumors derived from them arise from PCT. In comparison with PCT, basophilic cell tubules show slightly increased activities of all the enzymes studied. In basophilic cell tumors, markedly elevated hexokinase and pyruvate kinase activities are accompanied by a considerable reduction in the expression of GLUT2. GLUT1 expression is not found in basophilic cell tubules or PCT. Small basophilic cell tumors also do not express GLUT1, but GLUT1 is regularly expressed in several cell layers surrounding necrotic areas within large basophilic cell tumors. Our results indicate that increased glycolytic activity and reduced GLUT2 expression take place during the development of renal basophilic cell tumors.
This report describes a new method using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to visualize live viable tissue at the microscopic level. The visualization of the MTT stained tissue provides a metabolic assessment of tissue damage, and can be utilized as an extension of conventional hematoxylin-eosin (H & E) staining. In this report, several tissues were studied with MTT and H & E staining after incisions had been made by a variety of surgical dissecting instruments. A marked improvement in the detection of tissue damage was seen using MTT, regardless of how the damage was caused, i.e., physical, heat, or photon energy. In addition, a distinct zone of damage not noted on conventionally prepared and stained tissues is readily apparent. Thus MTT staining will have utility in both clinical and research studies, concerned with assessing the viability of tissues.
A thyroid carcinoma cell line, BHT-101, has been established in vitro from a metastatic lymph node deposit in a female patient with a non-hormone producing anaplastic, partly thyroglobulin- and thyroxine (T4)-positive papillary thyroid cancer. The cell population is heterogeneous, containing epithelial-like and fibroblast-like cells, and has a doubling time of 24 h. The cell line is polyploid with hypertetraploid predominance and the karyotype showed trisomies, tetrasomies, pentasomies as well as many marker chromosomes. The majority of the cells are negative or weakly positive for thyroglobulin and thyroxine and estrogen and progesterone receptors are present in the cells. BHT-101 cells produce tumours when injected into immunosuppressed CBA/Ca mice. The cells are sensitive to adriamycin, methotrexate and tamoxifen but not to methimazole (Favistan). The epithelial-like clone 1 and the fibroblast-like clone 3, isolated from the parental line, differed in drug sensitivity. This new cell line is suitable for studying the biology of thyroid carcinoma and for parallel in vivo and in vitro studies of drug activity against thyroid cancer.
The distribution of the interstitial collagens I, II and III was analyzed immunohistochemically in cartilage and bone samples from 32 patients with degenerative osteoarthrosis at various morphological stages. The alterations observed showed a very patchy, focal distribution demonstrating significant heterogeneity in the tissue reaction. In minor osteoarthrotic lesions a focal pericellular deposition of collagens III and I was seen, while the majority of the interterritorial matrix reacted exclusively with collagen II antibodies. These changes were first seen in the superficial cartilage layer. At the more advanced stages of osteoarthrosis, particularly when osteophytic bone spur formation was present, extensive changes in the expression of collagen types in the pericellular matrix was revealed with extensive and overlapping localization of collagens I, II and III in the whole cartilage. These observations support the suggestion that degenerative cartilage shows a collagen type "switch". In addition, it was demonstrated that the interterritorial cartilage matrix was still mainly composed of collagen II even in advanced lesions. These observations may explain some of the previous discrepancies reported.
The new cell line PaTu 8902 was established from a human pancreatic grade II adenocarcinoma of ductal origin. In early passages, cultured cells showed a high degree of heterogeneity in terms of their morphology and the number of chromosomes per cell. When transplanted subcutaneously into nude mice, these cells grew as tumors with a similar morphology and differentiation (grade II) to the primary tumor. In contrast, after prolonged cultivation, cells were more homogenous in terms of their morphology and number of chromosomes per cell, and the corresponding nude mouse xenografts were less differentiated (grade III). When cells from late passages were injected intravenously into nude mice, lung metastases occured after 3–4 weeks. In addition, tumor cells were found in the wall of the esophagus and in the pleural cavity, indicating a high metastatic potential for PaTu 8902 cells in nude mice.
Using the polymerase chain reaction (PCR) to examine the occurrence of bcl-2/JH joining produced by t(14;18) chromosomal translocation, amplified DNA was detected in 2 of 18 lymph nodes showing reactive lymphadenopathy. The PCR was repeated in these two lymph nodes using the same DNA samples, but no amplification was detected at the second attempt. Thus the amplified DNA was considered to be derived from one copy of joined bcl-2/JH in one cell, or from a few copies in a few clonal cells with the same joined bcl-2/JH. These results suggest that false joining of bcl-2/JH at the t(14;18) junction may occur in reactive lymph nodes.
The localization of alpha 1-antichymotrypsin (ACT) mRNA and ACT immunoreactivity (ACT-IR) were examined in 12 brains obtained at post-mortem from elderly patients, four of whom had Alzheimer's disease. A biotinylated oligonucleotide probe was used for in situ hybridization histochemistry and the relationship between the expression of both ACT mRNA and ACT-IR and the extent of beta protein or tau deposition were investigated. The extent of beta-plaques, tau-tangles, and ACT-IR were rated from (-) to (++). In brains without plaques and tangles, there were no detectable ACT mRNA signals in the gray matter, and those in the white matter were weak; in these brains, ACT-IR was generally weak. The brains with beta-plaques but no tangles showed weak ACT mRNA expression in astrocytes of both the gray and white matter; they also showed weak ACT-IR in the astrocytes. In the brains from patients with Alzheimer's disease with both plaques and tangles, ACT mRNA was expressed intensely in a majority of the astrocytes in the white and gray matter. Some senile plaques-associated astrocytes expressed ACT mRNA and ACT-IR was strong in the white matter astrocytes. ACT-IR and ACT mRNA expression in astrocytes was correlated with the extent of beta and tau depositions.
p68 is an inducible protein kinase which is believed to be an important factor in the regulation of both viral and cellular protein synthesis. We have produced a monoclonal antibody (TJ4C4) which specifically detects p68, and which can be used to detect this antigen in formalin-fixed, paraffin-embedded tissues. Because p68 plays an important role in cellular protein synthesis, we hypothesized that it may correlate with normal and neoplastic cellular differentiation. One hundred and seventy-seven head and neck squamous cell carcinoma specimens, representing 82 patients, were studied. The relative amount, frequency, and distribution of p68 expression were determined by microscopic evaluation of ABC immunoperoxidase-stained specimens. A spectrum of immunoreactivity was detected in 156 of 177 tumors, as well as within the normal squamous epithelium. Normal, actively proliferating cells, such as the basal layer of squamous epithelium, expressed comparatively little p68. Increased p68 expression was noted to parallel the morphologic features of cellular differentiation. In neoplastic tissue, p68 expression also increased with the degree of cellular differentiation. These data demonstrate that the expression of p68 parallels the degree of cellular differentiation in squamous cell carcinoma of the head and neck region, as well as within normal squamous mucosa. Therefore, p68 may provide an objective biologic measure of cellular differentiation which does not depend on morphologic features.
