Loss of pigment epithelium derived factor expression in glioma progression.

BACKGROUND: Pigment epithelium derived factor (PEDF) was first isolated from medium conditioned by human fetal retinal pigment epithelial cells. PEDF was detected in a broad range of human fetal and adult tissues including almost all brain areas. It can also inhibit the proliferation of cultured rat astrocytes. Recent studies have implicated PEDF in activities that are inhibitory to angiogenesis. AIMS: To investigate the expression of PEDF in gliomas to assess its "gliastatic" effects and its role in anti-angiogenesis. METHODS: PEDF mRNA values were measured by quantitative real time reverse transcription polymerase chain reaction (RT-PCR) analysis of normal brain tissue and tumour specimens from both low and high grade gliomas. In addition, immunohistochemical staining for PEDF and vascular endothelial growth factor (VEGF) was performed on 32 paraffin wax embedded glioma samples, 10 of them grade IV, 10 grade III, seven grade II, and five grade I. RESULTS: RT-PCR showed that PEDF mRNA values were 5.0 (p < 0.001) and 15.4 (p < 0.001) times higher in normal human brain specimens (n = 5) than in tumour tissue specimens of low grade glioma (grades I and II; n = 15) and high grade glioma (grades III and IV; n = 10), respectively. VEGF was strongly positive in 90% of grade IV, 70% of grade III, 43% of grade II, and 20% of grade I cases. In contrast, PEDF was positive in none of grade IV, 20% of grade III, 43% of grade II, and 60% of grade I tumours. There was an inverse correlation between VEGF and PEDF expression, and a lack of PEDF in advanced grade gliomas. CONCLUSIONS: It is possible that the absence of PEDF expression is a potent factor for the enhancement of angiogenesis in glioma.

Expression of a new surface membrane antigen (SQM1) in tumor cells cultured in media with different calcium ion levels.

Terminal differentiation is usually achieved in normal as well as transformed squamous epithelial cells when cultured. On the other hand, tumor cells at various differentiation stages and with different biological characteristics comprise the heterogeneous properties of tumors which have been one of the barriers to effective treatments. Recently, a surface membrane protein has been reported in squamous cell carcinomas of the head and neck, which is recognized by a murine monoclonal antibody, SQM1. This glycoprotein was further suggested to be related to squamous cell differentiation and intercellular adhesion. In a recent study, the esophageal carcinoma cells of EC/CUHK2 cell line were induced to various differentiation stages as evidenced by the increasing amount of intracellular desmosomes and tonofilaments and greater binding ratios of cytokeratin and involucrin antibodies than in those cells that maintained lower calcium ion concentrations. The expression of SQM1 antigen was found to increase in intensity when the tumor cells were cultured in moderate to high calcium ion levels for 10 to 15 hours when the differentiation patterns were beginning to appear. The intensity declined gradually thereafter. Thus SQM1 protein might be related to the stage when the cells started committing with squamous differentiation.

Establishment and characterization of two new cell lines derived from squamous cell carcinoma of the tongue in Chinese patients.

Two new cell lines derived from squamous cell carcinoma of the tongue, T1/CUHK and T2/CUHK, have been established in culture. Analysis of the morphology, ultrastructure, chromosome number, spheroid formation and immunohistochemical properties of the two cell lines demonstrated that they are both well characterized. T1/CUHK cells grew relatively faster than T2/CUHK cells. Both cell lines were tumorigenic after inoculation into made mice and showed positive reactivity with HPV 16 DNA probe. The reactivity of both cell lines with HPV 18 DNA probe was weak.

Fine structural observation of a nucleolar-nuclear matrix-lamina-intermediate filament system in transformed cells.

The nuclear matrix is a nonchromatin structure of the nucleus normally concealed by a much larger mass of chromatin. Several methods have been developed to remove chromatin from nucleus while preserving the underlying matrix architecture to some degree. The present study showed that after extraction of PtK2 cells and cervical carcinoma cells (CC3) with Triton X-100, ammonium sulfate and DNase, the nucleolar-nuclear matrix-intermediate filament (Nu-Nm-L-IF) network remained. The nucleolus was oval in shape and appeared as a fibrillar mass with an accentric dense fibrillar centre. This nucleolar skeleton was in direct connection with the nuclear matrix which in turn is connected with cytoplasmic intermediate filaments by lamins. It is concluded from these observations that the Nu-NM-L-IF system forms a continuous system which plays an important role in the maintenance of the nucleolar, nuclear and cytoplasmic integrity and cellular function.

Microwave-enhanced immunohistochemical staining of a formalin-sensitive antigen-laminin receptor.

Many potentially useful antigens have been difficult to detect in formalin-fixed, paraffin-embedded tissues. Recently a number of pathological and research laboratories have demonstrated that some antigens masked by formalin fixation could be restored to detectability by microwave heating. Previously, we were unable to demonstrate laminin receptor in cells processed by the routine fixation. Our results showed that microwave heating together with trypsin produced the best immunohistochemical staining for this receptor. Nevertheless, no significance was found in the levels of 67 kD LR in high and low metastatic tumor cell lines.

Biological characteristics of a newly established human cervical carcinoma cell line.

A new cell line, designated CC2/CUHK2, was established from a squamous carcinoma of the uterine cervix from a Chinese patient. The cell line grew well without interruption for over 12 months and over 90 passages. The doubling time of CC2/CUHK2 was 72 hours at passage 57. When seeded at a density of 1.5 x 10(3) cells per 25 cm2 tissues culture flask, the plating efficiency was 2.15%. Chromosome analysis showed a range of 30 to 130 with a modal number of 75. Immunoperoxidase staining of keratin showed positive reaction. Most of the CC2/CUHK2 cells showed weak nuclear staining of HPV capsid antigens with the ABC detection system. Analysis of the DNA samples extracted from the cervical cancer cells showed the presence of HPV type 16 DNA.

Electron microscopic analysis of the relationship between nuclear matrix stability and cell differentiation.

Two cell lines, the less differentiated CC2/CUHK2 and the more differentiated CC3/CUHKE3, were used to study the difference in nuclear matrix stability against DNase 1 digestion. The nuclear matrix was almost totally extracted when the CC3/CUHK3 cells were digested with 100 micrograms/ml DNase 1, while that of the CC2/CUHK2 cells was still present even when 200 micrograms/ml DNase 1 was used. It is suggested that more differentiated cells have a less stable nuclear matrix while the less differentiated ones have a more stable nuclear matrix. The same phenomenon was also observed in normal human and rat cervical epithelia. The nuclear matrix of the poorly differentiated basal cells was more stable than that of the more differentiated superficial cells. This cell differentiation stage dependent stability of the nuclear matrix is probably related to the nuclear activity and gene expression.

Effects of all-trans-retinoic acid on human esophageal carcinoma cells and their nuclear matrices.

Esophageal cancer is a common cancer among ethnic Chinese. The reported incidence of esophageal cancer has increased many fold in the past decades and is apparently still rising. Nuclear matrix forms the scaffold of the nucleus and participates in various nuclear functions. Cancer specific nuclear matrix proteins have been identified in several tumor systems. 2-D gel analysis shows the presence of novel nuclear matrix proteins in ATRA treated tumor cells and these proteins are cell line specific. Our preliminary study also shows ATRA altered the nuclear matrix density of tumor cells. The significance of these nuclear matrix proteins is discussed.

Effect of glucocorticoid hormone on nuclear matrix in cervical cancer cells in vitro.

Human papillomavirus (HPV) is one of the crucial factors in cervical carcinogenesis. High risk HPV16 prototype has been demonstrated in association with the nuclear matrix in a cervical carcinoma cell line(1,2). Nuclear matrix (NM) has been established as playing an important role in various nuclear activities as well as carcinogenic processes. Dexamethasone (DEX) (glucocorticoid hormone) inhibited the growth of CC2/CUHK2 cervical carcinoma cells with concurrent induction of epithelial cell differentiation. 2D- PAGE (IEF and NEPHGE) revealed alternations in NM protein composition. Further demonstration of changes in NM was evidenced by NuMA (a novel NM protein) labelling. The HPV16 E7 oncoprotein was shown to be reduced in total cellular protein as well as in NM protein fractions in response to DEX treatment, and this suppressed expression was confirmed by RT-PCR. Thus, it is suggested that dexamethasone can down-regulate the growth of cervical cancer cells and its induced changes in NM may be a cause of this suppression.

The association of HPV 16 DNA with specific nuclear matrix proteins of normal and cervical carcinoma cell.

Human papillomavirus type 16 (HPV 16) has been found to be integrated into the DNA of epithelial cells in most cervical cancers. The HPV16 DNA is bound to different nuclear matrix proteins in normal and cervical carcinoma cells. It has high affinity, for acidic proteins in cancer cells. The molecular weights of the acidic proteins are 200 kD, 186 kD and 67 kD. On the other hand, the viral DNA seemed to bind to higher molecular weight basic nuclear matrix proteins (250 kD, 150 kD) of normal cells. Further investigation of the functional roles of these nuclear matrix proteins may provide insight into the process of carcinogenesis of the cervix.

Electrophoretic analysis of nuclear matrix proteins in human hepatocellular carcinoma.

The nuclear matrix is the non-chromatin skeleton of the nucleus. This structure contributes to the shape of the nucleus and regulates various nuclear functions. In this study, nuclear matrix proteins of human normal liver, a liver cancer cell line, HepG2, and hepatocellular carcinomas (HCC) were investigated. Using high resolution two-dimensional polyacrylamide gel electrophoresis, the nuclear matrix proteins of 3 normal liver and 14 HCC were compared and contrasted. A high degree of similarity between normal liver, HepG2, and HCC nuclear matrix protein patterns was found. Two HCC specific nuclear matrix proteins were identified. Among these, one protein (HCC-1, Mr 62 kd, pI 5.3) appeared in all tumor samples and HCC-2 (Mr 33.25, pI 5.3-5.5) was present in 9/11 tumors, but absent in normal liver and HepG2. Our results indicate the presence of HCC specific nuclear matrix proteins. These matrix proteins may be used as markers for HCC.

Expression of nm23-H1 in human meningioma cells.

The expression of nm23-H1 has been demonstrated to be highly correlated with the metastatic potential of various tumors. In the present investigation, meningiomas of different pathological grades were used to study on their nm23-H1 expression. Immunohistochemistry showed that nm23-H1 was expressed mainly in the cytoplasm especially in the perinuclear region in explants under short-term culture. Western-blotting demonstrated the specific expression of nm23 protein in all tumor samples. The expression was also found to be sex-dependent on tumor progression in female, but not in male patients. RT-PCR results confirmed nm23-H1 expression was higher in benign tumors than in their normal counterpart. Our observations thus suggest that nm23-H1 may play an important role in the progression of meningiomas in female patients.

Ultrastructural localization of a new surface membrane antigen (SQM1) related to squamous differentiation.

Normal squamous epithelial cells readily undergo terminal differentiation in culture and are commonly used in differentiation studies. Several intracellular markers of squamous differentiation such as keratin, involucrin, transglutaminase and cholesterol sulfate have been well-studied and described by other workers. We have recently reported a surface membrane antigen in squamous carcinoma of the head and neck antigen in squamous carcinoma of the head and neck which is recognized by a murine monoclonal antibody SQMI. In this paper, we present our studies on the ultrastructural localization of SQMI antigen in cultured squamous epithelial cells using gold-labelled antibody. The cells studied included both normal and cancer cells at different degrees of differentiation. Under both transmission and scanning electron microscopy examination, the SQMI antigen was localized at the membrane surface of cultured cells, particularly at sites of cell-cell interdigitation. No association with desmosomal structure was observed in any of the specimens examined. There was however an association of SQMI antigen with microvilli of cell membrane. No non-specific cytoplasmic localization of SQMI antigen was observed. The intensity of SQM1 antigen revealed by gold-labelling appeared to have a positive correlation with the degree of differentiation of the cells in culture.

An immuno-electron microscopic study on the relationship between nuclear matrix and DNA in rat spermatocytes.

The nucleus of the mammalian spermatid undergoes a series of changes in its chromatin and nucleoprotein composition during transport from testis to epididymis. The sperm DNA is very tightly packaged by protamines instead of histones in somatic cells. However, the nuclear matrix and its association with DNA have not yet been definitively scrutinized with the electron microscope. The present study reveals that the protamine-depleted sperm nuclear matrix appears as a network of thick and thin filaments with glodular structures attached the these fibers. Monoclonal antibody to single- and doublestranded DNA was used to localize remnant DNA after extraction. By immunofluorescence microscopy, monoclonal antibody against DNA was localized outside the nucleus as a halo. Immuno-electron microscopy showed that gold particles were mainly associated with nuclear matrix surrounding the sperm head. Our results suggest a specific structural organization of sperm DNA with its matrix.

Ultrastructural study on the development of the rat [correction of mouse] spermatocyte nuclear matrix during epididymal maturation.

The rat sperm nucleus, after sequential extraction with detergents, nuclease and ammonium sulfate, consists of a skeletal structure that resembles the original nuclear shape. This chromatin-depleted skeleton is formed by thick and thin fibers as well as globular structures of different sizes. These fibers form anastomosis. The sperm nuclei obtained from testis and caput epididymis exhibits a loose fibrous network with thin fibers at the center. The entire nucleus of the sperm in the caudal epididymis is formed by a dense network of thick and thin fibers. These highly branched matrix fibers had diameters of 35 and 12 nm. It is concluded that the increase in density of the matrix fibers is related to the condensation of the chromatin in the maturation of the spermatozoa.

The growth and behaviour of pig retinal pigment epithelial cells in culture.

The retinal pigment epithelium contains melanin and lipofuscin. It is believed that in the in vivo system, the incomplete degradation of phagocytosed outer segment discs leads to the formation of lipofuscin. Our results showed that pig RPE cells can be successfully cultured using standard culture techniques without addition of specific growth factors. In this system, the autofluorescent material is formed mainly from the degradation of pigment granules. This culture system may provide an excellent model for studying of diseases related to the retina.

Fine structural observations on the human sperm nuclear matrix.

The nucleus of the mammalian sperm is formed after a series of morphological and biochemical changes during spermatogenesis. The human sperm nucleus, after sequential extraction with detergents, nuclease and ammonium sulfate, consists of a fibroskeletal structure which maintains the original nuclear shape. The chromatin-depleted skeleton is formed by thick and thin fibers as well as electron-dense patches of different sizes. These highly branched matrix fibers had average diameters of 35 and 12 nm. Polarization of the fibroskeletal structure is apparent and can be used as a good model to study the function of nuclear matrix in nuclear compartmentation in germ cells.