A unique role for Fyn in CNS myelination.

We analyzed the role of Fyn tyrosine kinase in CNS myelination by using fyn(-/-) null mutant mice, which express no Fyn protein. We found a severe myelin deficit in forebrain at all ages from 14 d to 1 year. The deficit was maximal at 1 month of age and was similar regardless of mouse strain background or whether it was determined by bulk isolation of myelin or by quantitation of myelin basic protein. To determine the cellular basis of the myelin deficit, we counted oligodendrocytes in tissue sections of mice expressing oligodendrocyte-targeted beta-galactosidase, and we used light and electron microscopy to examine the number and morphology of myelinated fibers and size of myelinated CNS structures. All of these parameters were reduced in fyn(-/-) mice. Unexpectedly, there were regional differences in the myelin deficit; in contrast to forebrain, fyn(-/-) cervical spinal cord exhibited no reduction in myelin content, number of oligodendrocytes, or number of myelinated fibers, nor was myelination delayed developmentally. We found that oligodendrocytes express Src, but there was no significant reduction of myelin content in null mutants lacking the Fyn-related kinases Src, Yes, or Lyn. Finally, we investigated the molecular features of Fyn that are required for myelination and found that a single amino acid substitution, which abolishes the tyrosine kinase activity of Fyn, resulted in a myelin deficit as great as that observed in the complete absence of Fyn protein. These results demonstrate that Fyn plays a unique role in myelination, one that requires its kinase activity.

Effect of glycoprotein and protein hormones on human meningioma cell proliferation in vitro.

Speculation that meningiomas are subject to female hormone influence is supported by their higher incidence in women and reports of exacerbation of symptoms during pregnancy and the luteal phase of the menstrual cycle. Previous reports have concentrated on the effects of the steroid hormones oestradiol and progesterone on meningioma proliferation. In this study we have investigated the roles of the glycoproteins LH, FSH and human chorionic gonadotrophin (hCG), and the protein hormones prolactin (PRL) and human placental lactogen (hPL) on the proliferation of human meningiomas in vitro. The three glycoproteins had an inhibitory effect on meningioma proliferation ranging from 5.0-50.0%, 10.0-63.0% and 2.4-34.0% at the highest concentrations of LH (25 mIU/ml), FSH (15 mIU/ml) and hCG (30 IU/ml) respectively. Cultures were also treated with PRL (100 and 200 ng/ml) and hPL (5 and 10 ng/ml) and the protein hormones had a stimulatory effect on cell proliferation of 12.0-55.5% and 11.4-73.6% when treated with 200 ng/ml PRL and 10 micrograms/ml hPL respectively. Our data suggest that increasing levels of the protein hormones PRL and hPL, falling levels of hCG and the absence of LH and FSH in the second and third trimesters of pregnancy may play a role in the acceleration of meningioma growth in these stages of pregnancy.

Interleukin-6 (IL-6) production and cell growth of cultured human ameningiomas:-interactions with interleukin-1 beta (IL-1 beta) and interleukin-4 (IL-4) in vitro.

We examined the production of interleukin (IL)-6 by human meningioma cells in vitro, and the effects of IL-1 beta and IL-4 on IL-6 production and meningioma cell growth. The histological classification of the tumours studied included transitional, syncytial, fibroblastic and atypical. All 10 meningiomas studied produced IL-6 (range 0.22-7.6 ng/ml/10(6) cells/24 h). Separate addition of IL-1 beta or IL-4 to cultures increased IL-6 production up to ten fold, and two to three fold, respectively. Growth studies with IL-6 indicated that this cytokine significantly increased terminal cell density at a concentration greater than 1 ng/ml in 60% of the meningioma cultures studied. IL-1 beta caused a significant decrease in the terminal cell density in 25% of the meningioma cultures studied whereas IL-4 had a tendency to significantly inhibit growth in 16.6% of the cultures. These data suggest that IL-6 production by meningiomas can be modified by other cytokines and secondly, that IL-6, IL-1 beta and IL-4 can modify growth in vitro and may act as autocrine factors in vivo. By further determining the cytokine profiles within meningiomas and their effects, a better understanding of meningioma growth characteristics may be obtained.

Constitutive co-expression of estrogen and progesterone receptor mRNA in human meningiomas by RT-PCR and response of in vitro cell cultures to steroid hormones.

Although it is well recognised that human meningiomas are rich in progesterone receptor (PgR), controversy has existed about the presence of the estrogen receptor (ER) in these tumours. We have investigated the presence of both ER and PgR in a series of 20 human meningiomas, spanning the main histological groups, using reverse transcription linked PCR (RT-PCR). Total RNA was extracted from whole tissues and reverse transcribed to yield cDNA. This was amplified using primers specifically designed to detect ER and PgR. All samples co-expressed ER and PgR mRNA, irrespective of tumour classification, patient age or sex. In general, transcripts for PgR appeared considerably stronger than those for ER, and although this was a purely qualitative study, it suggests increased expression of PgR. Addition of exogenous 17beta-estradiol or progesterone to meningioma cell cultures showed that 2/4 cultures responded to these steroids. Our results confirm that human meningiomas do express gene transcripts for ER, and that previous failures to detect ER in these tumours may be due to the lack of sensitivity of the techniques employed. However, these receptors may not be functional in all tumours.

Interleukin-3: a putative protective factor against breast cancer which is secreted by male but not female breast fibroblasts.

The enzyme 17 beta-hydroxysteroid dehydrogenase (17-HSD) is a key regulator of intracellular 17 beta-estradiol (E2), which is associated with breast cancer and is influenced by paracrine factors released by breast-cancer fibroblasts. Since the incidence of breast cancer is much higher in females than in males, we have used an in vitro cell culture system to investigate whether male fibroblasts may inhibit breast-cancer genesis by restricting the intracellular accumulation of E2. Fibroblasts were obtained from normal males and females undergoing reduction mammoplasty, and from females with benign or malignant breast lesions. Fibroblast-conditioned medium (CM) was incubated with the established breast-cancer cell line, MCF-7, and its effects on 17-HSD activity were assessed. CM (25% v/v) from male breast fibroblasts had a significant inhibitory effect on reductive 17-HSD, decreasing E2 production. This was in direct contrast to the effects of CM from female breast fibroblasts, which had a powerful stimulatory effect on reductive 17-HSD. RT-PCR allowing simultaneous detection of a range of cytokines was performed on each type of fibroblast. IL-3 mRNA was consistently detected in fibroblasts from male but not female breast tissue. Addition of rhIL-3 to cultures of MCF-7 caused a reduction in 17-HSD activity and addition of a polyclonal antibody directed against IL-3 to male CM completely reversed the inhibitory effects of CM. Thus, male breast fibroblasts may be responsible for secreting IL-3-like factors which, given the considerably lower incidence rates of breast cancer in men, may have a protective effect against breast cancer.

RT-PCR detection of cytokine transcripts in a series of cultured human meningiomas.

The expression of cytokine transcripts has been investigated in a series of cultured human meningiomas using reverse transcriptase linked polymerase chain reaction (RT-PCR), which allowed simultaneous analysis of a range of cytokines. The main histological subgroups of meningioma were investigated; these included transitional, fibroblastic, and syncytial as well as atypical meningiomas. Meningiomas from each of the different histological subgroups were subjected to a standard tissue culture regime. Total RNA was extracted from representative cultures and reverse-transcribed to yield cDNA. PCR was performed using oligonucleotide primers designed to detect interleukin (IL)-1 alpha/beta to IL-8, transforming growth factor (TGF)beta 1-3, tumour necrosis factor (TNF)alpha/beta, and interferon (IFN)gamma. Transcripts for IL-3, IL-6, IL-8, and TGF beta 3 were detected in all cultures. Transcripts for the three isomers of TGF beta were expressed in the transitional and fibroblastic meningioma cells. TGF beta 2 and TGF beta 3 transcripts were expressed in the syncytial and TGF beta 1 and TGF beta 3 in the atypical meningioma cells. IL-1 beta transcripts were expressed in fibroblastic and atypical cultures and TNF beta transcripts were expressed in syncytial and transitional cultures only. Transcripts for IL-1 alpha, IL-2, IL-4, IL-5, IL-7, TNF alpha, or IFN gamma were not detected in any of the meningioma cultures. This investigation using cells cultured from a small number of tumours from each of the classic histological subtypes suggests that there is a distinct pattern of cytokine mRNA expression linked with histological classification.