M. Suh

Clone pTB16 has been isolated by differential screening of a human glioma cDNA library. Northern blot analysis has shown that pTB16 expression is several times (greater than 11-fold) higher in gliomas than in a primitive neuroectodermal tumor. This observation was supported by in situ hybridization and extended to nine other gliomas. Expression was virtually absent in adenocarcinoma cells metastasized to brain. Malignant gliomas showed stronger hybridization than benign gliomas, while blood capillaries did not show hybridization. pTB16 mRNA was also shown to be expressed in established glioma cell lines and at high levels in epileptic foci, indicating that expression of the gene may be limited to certain cell types and that its upregulation is not merely a consequence of cellular proliferation. Nucleotide sequence analysis identified pTB16 as the human counterpart for rat testicular sulfated glycoprotein 2 (SGP-2), whose function in the reproductive system remains unknown. Although SGP-2 transcripts, and hence pTB16, were recently shown to be increased in neurodegenerative diseases such as scrapie in hamsters and Alzheimer disease in humans, our observations with brain tumors and epilepsy are suggestive of a role for pTB16 in neuropathologies in general and support the hypothesis of its involvement in tissue remodeling and cell death.

A leptomeningeal cell line (LM7) harbouring an unknown retrovirus was recently isolated from the cerebrospinal fluid of a patient with multiple sclerosis. However, spontaneous expression of the LM7 retrovirus in this primary culture is quite low. We present results showing that in vitro infection of LM7 cells with herpes simplex virus type 1 (HSV-1), but not that of control cells, results in (i) potent stimulation of the specific reverse transcriptase (RT) activity detected in the culture supernatant and (ii) co-expression of both typical HSV-1 virions and abundant retrovirus-like particles. Transfection of LM7 cells with plasmids expressing HSV-1 immediate early (IE) ICP0 and ICP4 proteins produced a similar enhancement of RT activity in culture supernatants with retrovirus-like particles being identifiable by electron microscopy. These effects were not observed with a plasmid expressing ICP27 or with the parental plasmid in LM7 cells, nor with any of these four plasmids in control cells. These results show that HSV IE trans-activating proteins strongly enhance the expression of the latent retrovirus present in LM7 cells. The possible role in vivo of herpesviruses as 'triggering' cofactors in the retrovirus hypothesis for multiple sclerosis aetiology is also discussed.

Sulfated glycoprotein-2 (SGP-2) gene expression seems to be constitutively expressed in a variety of tissues and organs, although levels of expression vary widely from one tissue to the other. SGP-2, also known as clusterin, has been reported to be expressed in the central nervous system (CNS). Some possible roles for brain SGP-2 have been postulated. In order to provide a substrate for a better understanding of the functions of this glycoprotein in the CNS, we investigated the detailed anatomical and cellular distribution of SGP-2 mRNA in the adult rat brain as well as the variation in its cellular expression after excitotoxin lesion. Transcripts for SGP-2 were found to be distributed throughout the rat CNS, although regional differences in their prevalence were readily observed. The ependymal lining of the ventricles showed the highest level of expression followed by various gray matter areas, some of which contained very intensively labeled cells. These cells were mostly found among several hypothalamic and brainstem nuclei, the habenular complex, as well as in the ventral horn of the spinal cord, which displayed striking hybridization signals over motoneurons. Occasional cells expressing high levels of SGP-2 transcripts were found in fiber tracts. Highly SGP-2 mRNA-positive resting glial cells were mainly located near the glial limitans and blood vessels. Two areas of relatively low constitutive SGP-2 mRNA expression are shown to produce strong hybridization signals 10 days after the local administration of the excitotoxin kainic acid. This overexpression of SGP-2 transcripts appears to involve GFAP-positive cells. Taken together, these results indicate that in the intact adult rat CNS, various cell populations, including neurons, constitutively express SGP-2 transcripts, whereas in the injured brain, reactive astrocytes become the major producers.

BHK cells infected with strain 17 syn+ (HSV-1) or HG52 (HSC-2) incorporated inorganic sulphate into polypeptides which co-migrated on SDS-polyacrylamide gels with virus-induced glycoproteins. The major sulphated glycoprotein was glycoprotein E. In addition, less-intense sulphated bands co-migrated with glycoprotein D and HSV-1 glycoprotein A/B/C. Sulphate label co-migrating with HSV-2 glycoprotein A/B/C was occasionally observed. We have investigated which sulphated polypeptides are excreted from infected cells. Major ones of apparent mol. wt. 32000, 34000 and 35000 were excreted from cells infected with syn+. In addition, polypeptides which migrated in the vicinity of glycoprotein D were often excreted from cells infected with either 17 syn+ or HG52. The 32K, 34K and 35K polypeptides were antigenically related to glycoprotein D and over 95% of the total amount synthesized was excreted. Analysis of intracellular sulphated polypeptides using intertypic recombinants mapped glycoprotein E to between 0.832 and 0.950 units of the HSV genome.

Syrian hamster embryo fibroblasts were oncogenically transformed by UV-inactivated Herpes simplex type 2. Eighteen clones were isolated shortly after transformation occurred. Two clones and their tumor derivatives were studied using several techniques. The karyotype analysis revealed different chromosome patterns in the two clones and a tendency toward hypodiploidy in the tumor derivatives. All of these cell lines were shown by molecular hybridization to contain 40% of the HSV-genome in several copies. The viral DNA sequence complexity was retained in the tumor derivatives, but a decrease in the copy number was observed. Viral RNA's were detected by in situ hybridization in all the lines that were tested. Viral antigens could be observed in these transformed cells by immunofluorescence. Finally, polypeptide analysis showed three differences between normal and transformed cells.