The measurement of AgNORs in human glioma cells.

Sixty gliomas obtained by neurosurgical resections were examined. Paraffin blocks were retrieved from pathological files of the Second Affiliated Hospital in Guangzhou Medical College. The methods of argyrophilic technique for AgNORs staining, and Image Analysis System for measurement of AgNORs were used. Six parameters, which included hcount, count, narea, agnrea, agpern and agperc were used to correlated well with histopathological grades (compared grade 2 & 3, grade 3 & 4, and grade 2 & 4, respectively). We concluded that AgNORs is useful in evaluating proliferative activity and assessing the malignancy of human gliomas. It may also be used as a target for anti-neoplastic drugs in the treatment of gliomas.

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.

Bcl-2 and Bax proteins are nuclear matrix associated proteins.

Bcl-2 and Bax proteins are implicated in the regulation of apoptosis. Nuclear matrix has been demonstrated to be associated with a vast array of functional and regulatory properties of cells. NuMA is one member of a class of nuclear matrix proteins that resides in both the nucleus and mitotic apparatus. The nuclear lamins appear to form a thin fibrous structure immediately underlying the inner nuclear membrane of eukaryotic cell nuclei. The association of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U343 was studied by confocal microscopy and Western blotting. Confocal microscopic images display that bcl-2 was localized at the peripheral of the nuclear matrix and Bax protein was located in the nuclear matrix. Western blotting detected a 26 kDa bcl-2 band and a specific band of Bax at around 66 kDa in nuclear matrix proteins. Our results suggest that bcl-2 and Bax proteins are nuclear matrix associated proteins.

Expression of bcl-2 and Bax in EGFR-antisense transfected and untransfected glioblastoma cells.

Glioblastomas are the most frequent and most malignant astrocytic gliomas. The epidermal growth factor receptor (EGFR) gene is the most frequently amplified and overexpressed in glioblastomas. The expression of bcl-2 and Bax in EGFR-antisense transfected and un-transfected glioblastoma cell line, U87E and U87V was studied by immunohistochemistry and western blotting. Our results show that the expression of Bax was stronger and bcl-2 was weaker in EGFR-antisense transfected cell line than the untransfected control. Bcl-2 and Bax genes are probably involved in the reduction of malignancy of glioblastoma cell caused by the introduction of EGFR-antisense into these tumor cells.

A study on the relationship between antisense EGFR cDNA fragments and nuclear matrix proteins in glioblastoma cells.

The association of antisense epidermal growth factor receptor (EGFR) cDNA fragments with nuclear matrix from EGFR-antisense transfected glioblastoma cell lines U343 and U87 was investigated. A 1015 bp DNA fragment (primer I-II) was amplified in both genomic DNA and nuclear matrix-associated DNA (NM DNA) from EGFR-antisense transfected glioblastoma cell lines U343E and U87E. Two different DNA fragments (940 bp and 110 bp) were amplified by primer I-III in both genomic DNA and NM DNA of U343E, while one 110 bp PCR product was shown with the same primer in both genomic DNA and NM DNA of U87E only. After EGFR-antisense transfection, the binding property of the 110 bp DNA fragment (primer IV-V) to nuclear matrix was not affected. Southwestern blotting demonstrated the presence of antisense EGFR cDNA binding nuclear matrix proteins. Our findings demonstrate that not only EGFR DNA is associated with nuclear matrix, but the transfected antisense EGFR cDNA also binds to nuclear matrix proteins. The nuclear matrix is most likely involved in the replication and transcription of antisense EGFR cDNA or hybridisation with sense mRNA in vitro.

Association of EGFR gene fragments with nuclear matrices in glioblastoma cell lines.

The association of epidermal growth factor receptor DNA fragments with nuclear matrix in glioblastoma cell lines was investigated. Two different DNA fragments (primer I-III, 940 bp and primer IV-V, 110 bp) were amplified in both genomic DNA and nuclear matrix-associated DNA. It was found that the 110 bp DNA fragment (primer IV-V) from a non-encoding region was more closely associated with the nuclear matrices of cell line U343, U373, A172, and T98 than with U87. The other DNA fragment (primer I-III) was found in both the genomic DNA and NM DNA from cell line of U343 and U87. Another long DNA fragment (primer I-II, 1015 bp) was not detected in the DNA of all cell lines. Our findings suggest that the attachment of the 110 bp DNA fragments to nuclear matrix may contribute to the growth and malignancy of glioblastoma.

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.

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.

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.

Intrafusal muscle fibers of duck muscle spindles.

Serial transverse paraffin sections of intrafusal muscle fibers of spindles from the extensor pollicis and the extensor digitorum communis of ducks show that only one type of intrafusal muscle fiber exists, based on the mid-equatorial nucleation pattern, diameter, and length. Although the overall range in fiber diameter at the mid-equatorial region is between 4.2-20.0 microns, the average caliber is 10.4 +/- 3.18 microns (S.D.) for spindles of the extensor pollicis and 9.3 +/- 2.11 microns (S.D.) for spindles of the extensor digitorum communis muscles. The range in spindle length for the extensor pollicis is 290-2,090 microns, average 1,120 +/- 569 microns (S.D.), and for the extensor digitorum communis 1,160-2,500 microns, average 1,745 +/- 367 microns (S.D.). The range in number of fibers per spindle for the extensor pollicis muscle is 5-12, average 8.2, and for the extensor digitorum muscle it is 1-11. In the extensor digitorum communis, there appear to be two groups, based on fiber number. Spindles of one group have a range of 5-11 fibers per spindle with an average of 7.2, whereas the second group has a range of 1-4 with an average of 2.7 fibers per spindle. The second group of spindles constitutes 52.5% of the 40 spindles studied, and of these 7.5% were monofibril spindles, 15.0% difibril, 17.5% trifibril, 12.5% quadrifibril spindles.

The sensory ending of duck muscle spindles.

Thin and thick types of muscle spindles have been demonstrated from the extensor pollicis (EP) and the extensor digitorum communis (EDC) wing muscles of the duck. The thin type shows a smaller capsule, innervated by a long and narrow sensory ending; the thick type consists of a larger capsule innervated by a truncated and wide sensory ending. Each spindle receives an afferent nerve fibre within the range of 2.1-8.4 microns diameter and the ratio between fibre and sensory ending is always 1:1. There is no secondary sensory ending in duck spindles. By electron microscopy, sensory terminals of different sizes may be seen to lie in depressions of various depths on intrafusal muscle fibres, usually without disturbing the external contour of the muscle fibre, and are covered externally by a rather thick layer of basement membrane.

The number, distribution and density of muscle spindles in two wing muscles of the domestic duck.

A study by serial longitudinal sections of two dorsal wing muscles of the duck, the extensor pollicis and the extensor digitorum communis, has shown that: (1) The extensor pollicis muscle shows a spindle content range of 19--27, mean 23.2, giving a spindle density of 76.7/g; the extensor digitorum communis muscle shows a spindle content range of 37--59, mean 47.6, and a spindle density of 119.0/g. (2) Spindles in the extensor pollicis muscle are randomly distributed, while those in the extensor digitorum muscle are located primarily in the proximal two thirds of the muscle. (3) The present data have been discussed in relation to previous observations of muscle spindles occurring in some avian and mammalian skeletal muscles.

The blood supply of duck muscle spindles.

The blood supply of duck muscle spindles from the extensor pollicis and the extensor digitorum comminis wing muscles has been studied by light and electron microscopy. Capillaries usually accompany the nerve bundle that innervates the spindle, approaching at an oblique angle around the midequatorial region. Capillaries may run for some distance along the surface of the outer capsule, or penetrate partially into the outer capsule and lie between layers of the capsule cell processes. Some capillaries also penetrate the outer capsule, running into the periaxial space and continuing further towards the polar region. They have been shown to be in close contact with intrafusal muscle fibres, from the juxta-equatorial to the polar region, but have not been encountered among sensory terminals in the mid-equatorial region.