American Journal ofPathology, Vol. 141, No. 3, September 1992 Copyight X) American Association of Pathologists
Transforming Growth Factor-r3 and Transforming Growth Factor -receptor Expression in Human Meningioma Cells Mahlon D. Johnson,*t Charles F. Federspiel,t Leslie 1. Gold,11 and Harold L. Mosest From the Division of Neuropathology,* and the Departments of Cell Biologyt and Preventive Medicine,* Vanderbilt
University Medical School, Nashville, Tennessee; and the Department of Pathology,11 New York University School of Medicine, New York, New York
The transforming growth factor-a (TGFI3) family in mammals includes three closely relatedpeptides that influence proliferation and numerous physiologic processes in most mesenchymal cells. In this study, Northern blots, immunohistochemistry, TGFP radioreceptor assays, TGFI receptor affinity labeling and 13H] thymidine incorporation were used to evaluate whether primary cell cultures of human meningiomas synthesize the three TGFF isoforms, bear TGFP receptors, and respond to TGF. Transcripts for TGFf3l and 2 were detected in the three cases analyzed Transforming growth factor-P1 immunoreactivity was detected in three of six cases, and TGFI2 and 3 immunoreactivity were detected in each case analyzed Media conditioned by cells cultured from six meningiomas also contained latent TGF4-like activity. Transforming growth factor-4 receptor crosslinking studies identified TGF3 binding sites corresponding to the type 1, type 2, and type 3 receptors on meningioma cells. Treatment with active TGF43I produced a statistically significant reduction in [3H] thymidine incorporation after stimulation with 10% fetal calf serum and epidermal growth factor in all six cases studied (Am JPathol 1992, 141:633-642)
Meningiomas constitute 25% of primary intracranial neoplasms in humans.1' 2 Although typically benign, perineural growth, growth at inoperable sites, and recurrence complicate traditional surgical management.1' 2 Resection is even less effective as a treatment for malignant variants that characteristically invade brain parenchyma and occasionally metastasize.'12 Development of alterna-
tive chemotherapy for these neoplasms has been hindered, in part, by the limited understanding of growth regulation in meningioma cells. Progesterone stimulation of meningioma growth has been suggested by numerous clinical studies3'4 and demonstrated in vitro.5 Other growth regulatory hormones, however, undoubtedly contribute to arachnoid neoplasia. Epidermal growth factor (EGF) and fibroblast growth factor stimulate human meningioma cell proliferation in vitro.-7 Recent demonstration of EGF in human CSF8 as well as EGF/TGFa receptors, platelet-derived growth factor (PDGF), PDGF receptors, and basic fibroblast growth factor (bFGF) mRNA in meningiomas9-13 raise the possibility that complex paracrine and autocrine stimulation by growth factors promote meningioma formation. Transforming growth factor 4-1 (TGFP1), TGFP2, and TGF433, another family of closely related peptides that regulate cell growth, may also participate. Transforming growth factor-p was originally identified as a peptide producing transformation of non-neoplastic cells in vitro, suggesting a role for this factor in oncogenesis.1"16 Subsequent studies, however, have described more diverse effects of the TGF,Bs on mesenchymal cell proliferation. Although stimulatory effects have been demonstrated in fibroblasts, osteoblasts, and smooth muscle myocytes,17-18 growth inhibition has been noted in epithelial myeloid and lymphoid cells." The recent demonstration of transcripts and immunoreactivity for TGF,B1, TGF,2, and TGF43 in murine and human meninges1921 (M. D. Johnson and H. L. Moses, unpublished observations) raises the possibility that TGFp1, TGFB2, or TGF,B3 influence normal arachnoid cell growth. Conceivably, loss or changes in this regulation contribute to neoplastic growth. The current study evaluated TGF,B1, TGF,B2, and TGF,B3 expression and TGF,B receptor presence in primary cultures established from six human meningiomas. Additional experiments asSupported by Veterans Administration Career Development Award (RA1793 MJ) and Merit Award (MJ). Accepted for publication March 3, 1992. Address reprint requests to Dr. Mahlon Johnson, Division of Neuropathology, Department of Pathology, Vanderbilt University Medical School, C3321 MCN, Nashville, TN 37232.
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sessed the effects of TGFp1 on quiescent, partially, and maximally growth-stimulated meningioma cells. Findings reported here suggest that meningiomas consistently synthesize and release all three isoforms of TGFP in a latent form and bear TGF, receptors. Treatment with TGF31 significantly reduced serum or EGF stimulation of proliferation in these cultures.
ma cells were
plated onto four-well microscope slides (Lab Tech) for 1 day in serum-free media, then fixed for 10 minutes in freshly prepared 4% paraformaldehyde in phosphate buffer. Subsequent analysis was performed using mouse monoclonal antibodies against human epithelial membrane antigen (EMA, DAKO, Carpinteria, CA), and vimentin (DAKO) and the biotin-avidinhorseradish peroxidase method.23 Epithelial membrane antigen and vimentin immunoreactivity are detected in approximately 50% and 18% of meningiomas, respectively.24
Tumor Characteristics Meningioma cell cultures were established from six patients after a frozen section diagnosis was made on adjacent tissue by one of us (MJ). Tumor 1 was a convexity transitional meningioma from a 43-year-old woman. Tumor 2 was an olfactory groove meningothelial meningioma from a 70-year-old woman. Tumor 3 was a meningothelial convexity meningioma of a 46-year-old woman. Tumor 4 was a frontal lobe fibroblastic meningioma from an 82-year-old woman. Tumor 5 was an orbital meningothelial meningioma from a 47-year-old man. Tumor 6 was a convexity fibroblastic meningioma in a 1 6-year-old, previously irradiated boy. Tissue was procured following procedures approved by the Vanderbilt University Institutional Review Board.
Meningioma Cell Cultures Small fragments of tumor were collected in supplemented Dulbecco's Modified Essential Media, then finely minced, using 22-gauge needles. Meningioma cells were seeded onto flasks and maintained in Dulbecco's modified essential media supplemented with 10% fetal calf serum (FCS), penicillin (50 units/ml), streptomycin (50 ,ug/ml), and Fungizone (Squibb, Princeton, NJ; 2.5 ,ug/ ml). Cells were fed twice weekly. Additional cells were plated onto sterile Lab Tech microscope slides (NUNC, Naperville, IL) for immunohistochemical studies.22
Meningioma Cell Characterization For ultrastructural analysis, meningioma cells grown in 35-mm dishes were fixed in 2% glutaraldehyde for 1 hour, gently scraped, then pelleted by centrifugation. The pellet was postfixed in 1% OS04 for 1 hour, then embedded in Epon (E. Fullam, Latham, NY). Thin sections were stained with uranyl acetate and lead citrate for ultrastructural analysis on a Philipps 300 electron microscope.22 For immunohistochemical characterization, meningio-
RNA Analysis by Northern Blot Polyadenylated mRNA was derived from cells of confluent meningioma cultures and TGFP-producing HT1080 fibrosarcoma cells (American Type Culture Collection, Rockville, MD) using oligo (dT) cellulose as described previously.25 Five micrograms mRNA was separated by electrophoresis in 1.2% agarose gels containing formaldehyde and transferred to nitrocellulose.26 Hybridizations with 1 x 106 cpm/ml of labeled probe were performed at 420C in 5 x SSC with 50% formamide, 250 ,ug/ml sheared salmon sperm DNA, hybridization, blots were washed in 1 x SSC with 0.1% sodium dodecyl sulfate (SDS) and 0.1 x SSC and 0.1% SDS for 45 minutes at 420C. Between hybridizations, the filters were stripped and reexposed to confirm removal of 32plabeled cDNA. cDNA probes for TGF13127 and cyclophilin,28 a constitutively expressed gene, were labeled using the random primer extension method.29 cRNA probes for TGF,B230 were transcribed using T7 polymerase.31 Because of loss of RNA after multiple probe strippings, analysis of TGF,33 expression was not undertaken.
Immunohistochemical Localization of TGFr,-3 The distribution of TGFP1-3 immunoreactivity was evaluated using rabbit polyclonal antibodies against TGFI1332 and avidin-biotin complex immunohistochemistry3 performed on 5-,u-thick sections of the formalin-fixed, paraffin-embedded tumors and meningioma cell cultures, as outlined previously.34 Polyclonal antibodies were produced in rabbits immunized with synthetic peptides of TGFP1, TGFP2, or TGF,B3. The TGFP1-3 antisera exhibit no cross-reactivity between the TGFP isoforms on Western blots.32 Preabsorption of each antiserum with the peptide against which they are directed ablates the antiserum's immunoreactivity in tissue sections.32 Addi-
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for 5 minutes in reducing buffer, and then electrophoresed on 5% to 7.5% polyacrylamide gels.
TGFI3 Radioreceptor Assay
[3H] Thymidine Incorporation
Near confluent cultures of early-passage meningioma cells in T75 flasks were washed, then incubated for 72 hours with 15 ml serum and growth factor-free 402 MCD,B media. After collection, a portion of each sample was acidified (pH 1.5) for 2 hours at room temperature, then restored to neutral pH. One-, 0.75-, 0.50-, 0.25-, and 0.1 25-ml samples of neutral and acid-activated conditioned media were evaluated in triplicate for TGFIcompeting activity by radioreceptor assay.3536 Briefly, 84A cells were plated at a density of 2 x 105 cell per well in six-well culture dishes and maintained in McCoy's 5a medium with 10% FCS for 1 day. The cells were subsequently washed three times with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), then rocked at room temperature for 1 hour with buffer (128 mmol/I [millimolar] NaCI, 5.0 mmol/I MgSO4, 1.2 mmol/I CaCI, 50.0 mmol/I Hepes, pH 7.5, 2 g/l BSA) before incubation with known concentrations of pure TGF,B2, neutral or acid-activated conditioned media, and 0.25 ng/ml 1251 TGFP2. 1251-TGFr31 and -132 can be used interchangeably in radioreceptor assays because both bind all three receptors with essentially equal affinity.35 After rocking at room temperature for 2 hours, the cells were washed three times with PBS containing 0.1% BSA, the solubilized for 10 minutes in PBS with 1% Triton X-100. Results were expressed as percentage 1251 TGFP2 binding after correction for nonspecific binding.36
In these studies, we evaluated the effects of TGF,B1 on quiescent, suboptimally, and optimally stimulated meningioma cells.6 Meningioma cells from M-1 through M-6 were plated at a concentration of approximately 1 x 104 cells/ml in 24-well dishes and grown to confluence in McCoy's medium containing 10% FCS. Cultures then were incubated for 24 hours with serum-free MCDB 402 media to achieve quiescence. In the first study, TGFI1 effects were tested on cultures optimally stimulated with 10% FCS. Duplicate cell cultures were treated with MCDB 402 media alone or MCDB 402 containing 10% FCS, 10% FCS and TGF,B1 (10 ng/ml), or TGFI1 (10 ng/ml) alone. At 12 hours, these treatments were replaced with [3H] thymidine 0.5 uCi/ml/ well. Cell proliferation was stopped either 12, 24, or 36 hours later by addition of 1 mol/l (molar) ascorbic acid. In a second experiment, TGF1l effects were evaluated in suboptimally stimulated duplicate cell cultures treated with MCDB 402 media alone or MCDB 402 containing EGF (10 ng/ml) and insulin (500 ng/ml; Collaborative Res., New Bedford, MA), EGF, insulin, and TGF,B1 (10 ng/ml; R&D, Minneapolis, MN) or TGF,1l (10 ng/ml) alone. At 12 hours, these treatments were replenished with identical media also containing [3H] thymidine 0.5 ,uCi/ml/well. Cell proliferation was stopped with 1 mol/l ascorbic acid as above. For scintillation counting, the cells were solubilized with 1 N NaOH per well and rocked for 30 minutes, then counted on a scintillation counter. For autoradiography, performed only on M-5 and M-6, cells were fixed in methanol (glacial acetic acid 3.1 followed by absolute methanol and 10% trichloroacetic acid for 10 minutes each. After washing in distilled H20, the cells were dried, then coated with NTB2 emulsion (Kodak, Rochester, NY) and exposed for 5 days. The emulsion was developed with Microdol (Kodak, Rochester, NY). Percentage of control (MCDB 402 alone) values were calculated using the logarithm of response for 10% FCS, 10% FCS and TGFP1, and TGF,1l alone divided by the log response for MCDB 402. These percentages of control values were calculated for each of the six tumors of each of the three times, yielding the 54 values given in the results section. Percentages of control values were calculated identically in the counterpart experiment, evaluating TGF,1l effects on EGF- and insulin-stimulated cells. Determinations for TGF,1l were not available for tumor, M-2; consequently there were 51 values.
tional characterization of these antisera
TGFI Receptor Crosslinking A TGFI receptor crosslinking assay was performed on cells from three meningiomas (M-4 through M-6) as described previously,37 with minor modifications. Briefly, cells were plated and grown to confluence in 100-mm dishes maintained in McCoy's media with 10% FCS as described above. Ice-cooled plates were washed twice with PBS then incubated rocking at 40C for 1 hour with binding buffer. Subsequently, buffer was replaced with binding buffer containing 2 ng/ml 1251 TGF1-1 without or with 200 ng/ml unlabeled TGFP-1. After rocking at 40C for 4 hours, the cells were washed with binding buffer without BSA then rocked at 4°C for 15 minutes with BSA-free binding buffer containing 0.25 mmol/l disuccinimidyl suberrate. After scraping in dissociation buffer, cell membranes were solubilized by rocking at 4°C for 30 minutes in 1% Triton X-1 00. Extracts then were heated to 1 00°C
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Statistical analysis for the results was conducted using the GLM procedures for analysis of variance, with contrasts provided through SAS Statistical Software.3e In addition, nonparametric tests were used to analyze the effects of TGFIil when given alone.
Cytologic, Ultrastructural, and Immunohistochemical Characterization Each meningioma culture was populated by large polygonal cells with irregular, often interdigitating borders and central oval nuclei with multiple chromocenters. Whorl formation was frequently seen. Typically, by passage 5 to 6, senescence occurred, with cells exhibiting markedly decreased proliferation-expanded cytoplasm and vacuolation of the nucleus. These findings are characteristic of meningioma cell cultures.22 Ultrastructurally, interdigitation of cell membranes and desmosomes were identified in each meningioma culture. Extensive or moderate EMA immunoreactivity and extensive vimentin immunostaining were detected in the cells cultured from the meningiomas (data not shown).
Northern Blot Analysis The 2.3- to 2.5-Kb TGF,1 transcript was detected in 5 ,ug poly A + RNA from the control cells and each of the three meningiomas tested (M-2, M-4, M-5) (Figure 1). In addition, the 5.1 -Kb TGF,2 transcript was also detected in HT 1080 cells and each of the meningiomas (Figure 1). The 1.0-Kb cyclophilin transcript was used as a control for RNA loading and integrity.
TGFP Immunohistochemistry Immunoreactivity detected for TGFP1, TGF02, and TGFPi3 in formalin-fixed meningioma tissue and meningioma cells is listed in Table 1. Transforming growth factor beta-1 immunostaining was detected in tissue sections from two of six meningiomas examined. Immunoreactivity was confined to the cytoplasm of tumor cells and was not seen in the blood vessels or dura. In cultured cells, extensive TGF,B cytoplasmic immunoreactivity was seen in M-2 and scattered immunostaining was detected in M4. Transforming growth factor P-1 immunostaining was confined to the cytoplasm, often distributed in a discrete pattern suggesting Golgi localization. Loss of the
I 'I4 3 Figure 1. TGF1 and 32 mRNA in cultured human meningioma cells. Each lane contains approximately 5 pg poly (A) + RIVAfirom HT1080 fibrosarcoma cells known to express TGF1, TGF[2, and TGF133 (lane 1), TGF producing SW620 adenocarcinoma cells (lane 2) and H9 lymphoblasts (lane 3), M-2 (lane 4), M-4 (lane 5) and M-5 (lane 6). Filters were hybridized with 32-labeled cyclophilin cDNA probe (A), 32P-labeled TGF131 cDNA (B), and 32Plabeled TGMFf2 riboprobe (C).
M-3 meningioma cells precluded immunocytochemical analysis of the other positive tumor. Transforming growth factor beta-2 immunoreactivity was detected in tissue sections from each of the six tumors and in cells cultured from five of the six tumors. In the tumors, TGFI2 immunostaining was distributed primarily in the cytoplasm of tumor cells. Scattered immunoreactivity also was detected in the muscularis of large arterioles in one tumor. No distinct immunostaining was detected in the dura. In cultured meningioma cells, extensive immunostaining was seen in M-1 and M-2, and scattered immunoreactivity was detected in cells from M-4-M-6 confined to the cytoplasm, in a pattern similar to that of TGF,Bl (Figure 2). Transforming growth factor P-3 immunostaining was seen also in tumor sections from each of the six meningiomas and in the cultured meningioma cells from Table 1. TGF Immunoreactivity in Cultured Meningioma Cells and Meningiomas Meningioma TGFoI TGFP2 M-1 tumor + cells + M-2 tumor + + cells + + M-3 tumor + rare + cells nd nd M-4 tumor cells + + M-5 tumor + cells + M-6 tumor + cells +
TGF,3 + + + + + nd + + + + + +
+ = positive immunostaining in tumor cells; - = no distinct immunostaining in tumor cells; nd = not done (no cells available).
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Figure 2. a: TGF2 immunoreactivity in cellsfrom meningioma culture M-1. Distinct cytoplasmic immunostaining (red) waspresent in many cells. AEC chromagen, Magnification, x240. b: TGF2 immunoreactivity in meningioma M-1. Cytoplasmic immunoreactivity was seen in cellular whorls throughout the tumor AEC chromagen magnification, X258
each of the five cases evaluated. In the tumor sections, moderate or extensive immunoreactivity was seen in tumor cells. Immunostaining was also seen in the media of one tumor arteriole and in dural arterioles. No distinct immunoreactivity was detected in the dura. In meningioma cells maintained in culture, TGF,3 immunoreactivity was also extensive in M-1 and M-2 and scattered-in, M-4 through M-6. In cultured cells, immunoreactivity was again confined to the cytoplasm, often perinuclear in distribution.
TGF, Radioreceptor Assays Neutral conditioned media from each meningioma contained no significant TGFO competing activity, suggesting that the meningioma cells do not synthesize TGFB1 -3 in an active form. Acid activation of the media from each meningioma culture, however, produced significant dose-dependent competition with 125TGF12, indicating that meningioma cultures synthesize latent TGFps. Approximately 9 ng/ml (M-1), 8 ng/ml (M-2), 2 ng/ml (M-3), 6 ng/ml (M4), 1 ng/ml (M-5), and 1 ng/ml (M-6) were secreted by late passage (P4-5) meningioma cell cultures (Figure 3).
TGF, from binding to the 85 kd (type 11) and 250 to 300 kd (type 111) receptors, as demonstrated by the disappearance of these bands (Figure 4).
3H-Thymidine Incorporation Data from the study evaluating TGFi1 effects on [3H] thymidine incorporation after stimulation with 10% FCS are graphed in Figure 5. Incubation with serum-free MCDB 402 medium was associated with a slight increase in [3H] thymidine incorporation 36 and 48 hours after addition, indicating continued viability of cells in serum-free media. Treatment with 10% FCS significantly increased [3H] thymidine incorporation in each meningioma culture at each time point. Transforming growth factor beta-1 (10 ng/ml) in serum-free media had no effect on 100
m m L._
TGF,3 Receptor Crosslinking
In the three meningioma cultures tested, affinity crosslinked bands were identified at the 45- to 60-kd, 70- to 90-kd, and the 250-300-kd regions of the gels. These bands correspond to the type 1, type 2, and type 3 receptors, respectively.3435 Competitive inhibition using 200 ng/ml unlabeled TGFI31 significantly reduced the intensity of the 65-kd band and completely displaced 1251
Condi'lo-nd Modium Added -ul
Figure 3. TGF,3 binding competing activity in meningioma cell conditioned media. Increasing volumes of acid-treated conditioned mediafrom M-1 (--- -), M-2 (--), M-4 ( ), M-5 ( . *), and M-6 (-.-) as well as untreated conditioned media from M-4 (--), M-5 (--), and M-6 [-, (M-6 over laps M-5)I were assayed in triplicate.
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Figure 4. Affinity labeling of meningioma cells with "25 TGF;11. Confluent monolayers of meningiomas M-4, M-5, and M-6 were incubated with 2 ng of 1'5I-TGF31, M-6 was also co-incubated with excess (200 ng) unlabeled TGF1.
M-2, M4, M-5, and M-6, but produced a statistically insignificant attenuation in [3H] thymidine incorporation in cells from M-1 at 24, 36, and 48 hours and M-3 at 24 hours relative to controls. In contrast, FCS-stimulated cells treated with TGF,1 consistently exhibited a significant reduction in [3H] thymidine incorporation compared with FCS alone in all six meningiomas at each time point. Autoradiography to determine percent labeled nuclei on M-5 and M-6 showed similar results (data not shown). Findings from the study evaluating TGFp1 effects on [3H] thymidine incorporation after suboptimal stimulation with EGF/lnsulin are illustrated in Figure 6. Incubation with serum-free media was again associated with a slight increase in [3H] thymidine incorporation. Administration of EGF/insulin produced a significant increase in [3H] thymidine incorporation in all six meningiomas at all time points. Transforming growth factor beta-1 alone resulted in an insignificant decrease in incorporation in cells from M-1, M-3, and M-5 at 24, 36, and 48 hours. Treatment of cells with EGF/insulin and TGF1 significantly reduced EGF/insulin's stimulatory effect in all six meningiomas at each time point. Within any given culture, differences observed at the different treatment times were not statistically significant.
The presence of mRNA and protein of all three mammalian isoforms have been demonstrated in the mouse1 921 and in human leptomeninges (MD Johnson and HL Moses, unpublished observations). These observations raised the possibility that neoplasms of the meninges might synthesize, secrete, and respond to members of the TGF,B family in an autocrine fashion. Findings presented here indicate that meningioma cells do, in fact, synthesize and secrete all three TGF, isoforms. Although the extent of the TGFP1, TGF,B2, and TGF,3 expression in various benign and malignant subtypes of meningiomas is not known, recent detection of TGFp1 in tumor tissue from several meningiomas,39 and the present data suggest consistent expression in these mesenchymal tumors. Moreover a recent report that acid/ethanol extracts of one human meningioma exhibited TGFP-like activity on NRK49F fibroblasts also may present latent TGF, activated by the extraction procedure.40 The extent and pattern of expression of all three TGFIs in meningiomas has not been reported previously. Takahashi et a139 recently detected TGFo1 transcripts in five tumors of unspecified subtype. Analysis of TGF,2 and TGF,B3 were not performed, however. Findings presented here suggest that at least meningiothelial and fibroblastic meningiomas produce all three isoforms of TGF,B. Moreover, TGFI2 or TGF,3 may represent the more abundantly synthesized isoforms. Although the biologic effects of TGF,B2 and TGF,B3 on meningioma cells have not been reported, all three TGFP isoforms bind the same surface receptors. Moreover, all three isoforms appear to produce similar biological effects in other cell types in culture.41 Ongoing analysis comparing expression of the three isoforms in meningioma subtypes may clarify which species predominate. Nonetheless the current observations suggest that meningiomas, like their arachnoid precursors, synthesize multiple members of the TGF3 family. Comparison of TGF,B-competing activity in neutral and acid-activated conditioned media suggests that meningioma cells secrete the latent, TGFP precursor in concentrations ranging from 1 to 10 ng/ml. Similar findings have been reported in other epithelial cells and fibroblasts that also synthesize and secrete TGF, in its latent form.4243 Because latent TGF3 does not bind to the TGF, receptors, it is unlikely that the secreted product exerts autocrine regulation of meningioma growth perse. This inability to activate TGFps may represent one facet of defective growth regulation permitting meningioma formation. Transforming growth factor P-i's role, if any, in regulation of meningioma cell growth awaits elucidation. Autocrine TGF, stimulation of cell growth, however, might
TGFp1-3 and Receptors in Meningioma Cells 639 AJP September 1992, Vol. 141, No. 3
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Figure 6. Effects of TGFf13 on [3H] thymidine incorporation in quiescent and EGF/Insulin treated meningioma cellsfrom six meningiomas. Thymidine incorporation in cells from each tumor is expressed as a percentage of the appropriate control 12, 24 or 36 h after addition of [3-'H thymidine. The data were evaluated by analysis of variance. EGF/Insulin ( ), EGF/Insulin and TGB3 (--), TGF31 alone (---).
type on several mesenchymal cells (ie, fibroblasts, chondroblasts, and osteoblasts), but is not present on epithelial cells.`051 Hence, its limited expression (relative to functional type and 11 receptors) on meningioma cells may be another manifestation of the meningiomas mixed mesenchymal/epithelial phenotype. Accumulating evidence suggests that the type III receptor/beta-glycan is a cell surface protein without signal transducing properties.51 Its role, if any, in meningioma cell physiology awaits elucidation. Transforming growth factor beta isoforms could influence a number of other cellular processes in meningiomas. Transforming growth factor beta-1 stimulates synthesis of fibronectin and collagen while inhibiting synthesis of collagenase.5254 Thus TGF,s may be an important modulator of fibronectin and collagen synthesis by meningioma cells.1' 22 Angiogenic properties of TGF,1, including stimulation of endothelial cell proliferation in vivo and induction of capillary formation might contribute to the formation and infiltration of leptomeningeal blood vessel into these highly vascularized neoplasms.47'48 In addition, the ability of TGF,B1 to stimulate osteoblasts and influence bone remodeling raises the possibility that TGFI family members modulate the hyperostosis that frequently occurs in the skull overlying meningiomas.1,1844 Future studies may clarify the role of TGF,B isoforms, if any, in mediating these changes.
Collectively, these data suggest that meningiomas synthesize one or more isoforms of TGF, that, when activated, inhibit meningioma cell proliferation. These findings raise the possibility that local infusion of degradation-resistant TGF,B agonists, endogenous activators of TGF,B such as plasmin,43 or other agents that remove the latency-associated peptide might slow tumor growth or reduce the incidence of recurrence.
References 1. Russell DS, Rubinstein U: Pathology of Tumors of the Nervous System. Fifth Edition. Baltimore, Williams & Wilkins, 1989, pp 449-532 2. Burger PC, Scheithauer BW, Vogel FS: Surgical Pathology of the Nervous System and its Coverings. 3rd ed. New York: Wiley, 1991, pp 67-93 3. Bickerstaff ER, Small JM, Guest IA: The relapsing course of certain meningiomas in relation to pregnancy and menstruation. J Neurol Neurosurg Psychiatry 1958, 21:89-91 4. Chaudhuri PK, Wallenburg HCS: Brain tumours and pregnancy. Eur J Obstet Gynecol Reprod Biol 1980,11:109-114 5. Jay JR, MacLaughlin DT, Riley KR, Martuza RL: Modulation of meningioma cell growth by sex steroid hormones in vitro. J Neurosurg 1975, 62:757-762 6. Weisman AS, Villemure JG, Kelly PA: Regulation of DNA synthesis and growth of cells derived from primary human meningiomas. Cancer Res 1986, 46:2545-2550
7. Koper JW, Foekens JA, Braakman R, Lamberts SWJ: Effects of progesterone on the response to epidermal growth factor and other growth factors in cultured human meningioma cells. Cancer 1990, 46:2545-2550 8. Hirata Y, Uchihashi M, Nakajma H, Fujita T, Matsukura S: Presence of human epidermal growth factor in human cerebrospinal fluid. J Clin Endocrinol Metab 1982, 55:11741177 9. Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, Whittle N, Waterfield MD, Ullrich A, Schlessinger J: Amplification enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumors of glial origin. Nature 1985, 313:144-147 10. Weisman AS, Raguet SS, Kelly PA. Characterization of the epidermal growth factor receptor in human meningioma. Cancer Res 1987, 47:2172-2176 11. Reubi JC, Horisberger U, Lang W, Koper VW, Braakman R, Lamberts SWJ: Coincidence of EGF receptors and somatostatin receptors in meningiomas but inverse differentiationdependent relationship in glial tumors. Am J Pathol 1989,
134:337-344 12. Maxwell M, Galanopoulos T, Hedley-Whyte ET, Black P McL, Antoniades HN: Human meningiomas co-express platelet-derived growth factor (PDGF) and PDGF-receptor genes and their protein products. Int J Cancer 1990, 46:1621 13. Kazumoto K, Tamura M, Hoshino H, Yuasa Y: Enhanced expression of the sis and c-myc oncogenes in human meningiomas. J Neurosurg 1990, 72:786-791 14. Moses HL, Branum EB, Proper JA, Robinson RA: Transforming growth factor production by chemically transformed cells. Cancer Res 1981, 41:2842-2848 15. Roberts AB, Anzano MA, Lamb LC, Smith JM, Sporn MB: New class of transforming growth factors potentiated by epidermal growth factor: Isolation from nonneoplastic tissues. Proc Natl Acad Sci USA 1981, 78:5339-5343 16. Shipley, GD, Tucker RF, Moses HL: Type B-transforming growth factor/growth inhibitor stimulates entry into S phase in monolayer cultures of AKR-2B cells after a prolonged prereplicative interval. Proc Natl Acad Sci USA 1985, 82:41474151 17. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross: TGF-Beta induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 1990, 63:515-524 18. Moses HL, Yang EY, Pietenpol JA: TGF-4 stimulation and inhibition of cell proliferation: New mechanistic insights. Cell 1990, 63:245-247 19. Pelton RW, Nomura S, Moses HL, Hogan BLM: Expression of transforming growth factor beta-2 RNA during murine embryogenesis. Development 1989, 106:759-767 20. Pelton RW, Dickinson ME, Moses HL, Hogan BLM: In situ hybridization analysis of TGFp1 and P2. Development 1990, 110:609-620 21. Heine VI, Munoy EF, Flanders KC, Ellingsworth LR, Lam PHY, Thompson NL, Roberts AB, Spom MB: Role of transforming growth factor-p in the development of the mouse embryo. J Cell Biol 1987,105:2861-2876
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22. Rutka JT, Giblin J, Dougherty DV, McCulloch JR, DeArmond SV, Rosenblum ML: An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma, cultures. J Neuropathol Exp Neurol 1986, 45:285-303 23. Warnke R, Levy R: Detection of T and B cell antigens with hybridoma monoclonal antibodies: A biotin-avidinhorseradish peroxidase method. J Histochem Cytochem 1980, 28:771-776 24. Meis JM, Ordoney NG, Bruner JM: Meningioma: An immunohistochemical study of 50 cases. Arch Pathol Lab Med 1986,10:934-940 25. Aviv H, Leder P: Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 1972, 69:14081412 26. Thomas PS: Hybridization of denatured RNA transferred or dotted to nitrocellulose paper, Methods in Enzymology. Edited by R Wu, L Grossman, K Moldave. Vol 100. New York, Academic Press, 1987, pp 255-266 27. Derynck R, Jarrett JA, Chen EY, Goeddel DV: The murine transforming growth factor-beta precursor. J Biol Chem 1986, 261:4377-4379 28. Danielson PE, Forss-Petter S, Brow MA, Calavetta L, Douglass J, Milner RJ, Sutcliffe JG: p1 B15: A cDNA clone of the rat mRNA encoding cyclophilin. DNA 1988, 7:261-267 29. Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983, 132:6-13 30. Miller DA, Lee A, Pelton RW, Chen EY, Moses HL, Derynck R: Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissue and embryos. Mol Endocrinol 1989, 3:1108-1114 31. Cox KH, DeLeon DV, Angerer LM, Angerer RC: Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 1984,101:485-502 32. Pelton RW, Saxena B, Jones M, Moses HL, Gold LI: Immunohistochemical localization of TGF,B1, TGFI2 and TGF,3 proteins in the mouse embryo. J Cell Biol 1991, 115:10911105 33. Hsu S-M, Raine L: The use of avidin-biotin-peroxidase complex (ABC) in diagnostic and research pathology, Advances in Immunochemistry. Edited by RA DeLellis. New York, Mason, 1984, pp 31-42 34. Pelton RW, Johnson MD, Saxena B, Perkett BA, Moses HL, Gold LI: Expression of TGF31, 132, and 133 mRNA and protein in the murine lung. Am J Respir Dis Cell Mol Biol 1991, 5:522-530 35. Lyons RM, Miller DA, Graycar JL, Moses HL, Derynck R: Differential binding of transforming growth factor -,11, -32 and -133 by fibroblasts and epithelial cells measured by affinity cross-linking of cell surface receptors. Mol Endocrinol 1991, 5:1887-1896 36. Tucker RF, Branum EL, Shipley GD, Ryan RJ, Moses HL: Specific binding to cultured cells of 1251-labeled transforming growth factor-type 13 from human platelets. Proc Natl Acad Sci USA 1989, 81:6757-6761 37. Massaque J, Like B: Cellular receptors for type B transforming growth factor, ligand binding and affinity labeling in hu-
Johnson et al
AJP September 1992, Vol. 141, No. 3
man and rodent cell lines. J Biol Chem 1985, 260:26362645 Statistical Users Guide: Statistics. Version 6. Cary, NC: SAS Institute, 1985 Takahashi JA, Mori H, Fukumoto M, Igarashi K, Jaye M, Oda Y, Kikuchi H, Hatanaka M: Gene expression of fibroblast growth factors in human gliomas and meningiomas: Demonstration of cellular source of basic fibroblast growth factor mRNA and peptide in tumor tissues. Proc NatI Acad Sci USA 1990, 87:5710-5714 Nitta T, Sato K, Okumura K: Transforming growth factor (TGF),B-like activity of intracranial meningioma and its effect on cell growth. J Neurol Sci 1991, 101:19-23 Graycar JL, Miller DA, Arrick BA, Lyons RM, Moses HL, Derynck R: Human transforming growth factor 133: Recombinant expression, purification and biological activities in comparison with transforming growth factors-beta 1 and beta 2. Mol Endocrinol 1989, 3:1977-1986 Lawrence DA, Pircher R, Krycena-Martinerie C, Jullien P: Normal embryo fibroblasts release transforming growth factors in a latent form. J Cell Physiol 1984, 121:184-188 Lyons RM, Gentry LE, Purchio AF, Moses HL: Mechanism of activation of latent recombinant transforming growth factor 11 by plasmin, J Cell Biol 1990,110:1361-1367 Bamard JA, Lyons RM, Moses HL: The cell biology of transforming growth factor 13. Biochim Biophys Acta 1990,1032: 79-87 Shipley GD, Pittelkow MR, Wille JJ, Scott RE, Moses HL: Reversible inhibition of normal human prokeratinocyte proliferation by type 13 transforming growth factor-growth inhibitor in serum-free medium. Cancer Res 1986, 46:2068-2071 Beck SL, Chin TL, Hiraboyashi SE, Deguzman L, Lee WP, McFatridge LL, Xu Y, Bates RL, Ammann AJ: Accelerated healing of ulcer wounds in the rabbit ear by recombinant
human transforming growth factor-31. Growth Factors 1990, 2:273-282 Baird A, Durkin T: Inhibition of endothelial cell proliferation by type-beta transforming growth factor: Interactions with acidic and basic fibroblast growth factors. Biochem Biophys Res Commun 1986,138:476-482 Roberts AB, Spom MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine VI, Liotta LA, Falanga V, Kehil, JH, Fauci AS: Transforming growth factor-beta: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci 1986, 83:41674171 Boyd FT, Massague J: Growth inhibitory response to TGF-13 linked to expression of a 53 kDa cell surface TGF-,B receptor. J Biol Chem 1989, 264:2272-2278 Andres JL, Stanley K, Cheifety S, Massaque J. Membraneanchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth-P. J Cell Biol 1989,109:3137-3145 Segarini PR, Rosen DM, Seyedin SM: Binding of transforming growth factor ,B to cell surface proteins varies with cell type. Mol Endocrinol 1989, 3:261-272 Ignotz RA, Massaque J: Transforming growth factor 13 stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 1986, 261:4337-4345 Ignotz RA, Takeshi E, Massaque J: Regulation of fibronectin and type collagen nRNA levels by transforming growth factor-P. J Biol Chem 1987, 262:6443-6446 Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty J, Angel P, Heath JK: Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987, 6:1889-1904