Ubiquitin in normal, reactive and neoplastic human astrocytes

Brain Reaearch. 50(/(1989) 343-351

343

Elsevier BRES 14905

Ubiquitin in normal, reactive and neoplastic human astrocytes P a m e l a G. G a l l o w a y ~ a n d M a t t J. L i k a v e c 2 I Deparlment of Pathology, Children's Hospital Medical Center ()]'Akron, Akron, OH 44308, (U.S.A.) and -'Department of Surgery, Case Western Reserve University, Cleveland Metropolitan General Hospital, Cleveland, OH 44109 (U.S.A.)

(Accepted 28 March 1989) Key words: Astrocyte; Astrocytoma; Central nervous system neoplasia; Glioblastoma multilk)rme:

Immunocytochemistry; Ubiquitin

Ubiquitin, a protein important in regulating non-lysosomal proteolysis, has previously been shown to be present in cytoskeletal inclusions of the neurodegenerative diseases. Its role in other pathological processes of the central nervous system, such as neoplastic transformation of cells, is not known. The astrocytoma, a tumor of complex biology derived from the astrocyte, is the most common primary parenchymal human brain tumor in both children and adults. Until recently, ubiquitin was not known to form stable conjugates in cells. We have shown using immunocytochemistry on sections of astrocytomas that both glial fibrillary acidic protein (GFAP) (the major intermediate filament protein present in normal, reactive and neoplastic astrocytes) and ubiquitin are simultaneously present in the cytoplasm and cell processes of tumor cells. The presence of ubiquitin and GFAP was also found in astrocytoma cells in short- and long-term culture, and confirmed by immunostaining of Nots of tumor homogenates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. INTRODUCTION In the central nervous system, the most c o m m o n primary p a r e n c h y m a l t u m o r in both children and adults is the a s t r o c y t o m a 37. The biology of the a s t r o c y t o m a is complicated. A l t h o u g h the parent cell is the astrocyte, astrocytomas vary in degree of malignancy according to site within the neuraxis and age of the patient. The degree of malignancy varies from low-grade a s t r o c y t o m a (least malignant), to anaplastic astrocytoma, to the glioblastoma multiforme (most malignant) 3s. Unfortunately, little is known about the basic biology of astrocytomas. Many efforts in understanding the neoplastic transformation of cells are directed toward analyzing biochemical differences between the normal and the neoplastic counterpart. By examining proteins involved in cellular control mechanisms in both t u m o r cells and the normal parent cell, differences may be found that are related to the underlying neoplastic transformation. One such key protein is ubiquitin,

an 8.6-kDa protein whose amino acid sequence shows only two substitutions from yeast to man '~. This e x t r e m e conservation of structure indicates that ubiquitin plays a vital role in cell function. Ubiquitin normally plays a central role in non-lysosomal protein degradation; proteins c o n j u g a t e d to ubiquitin are rapidly b r o k e n down by A T P - d e p e n d e n t proteasesl3'L4'L7 The role of ubiquitin in pathological processes has only been very recently investigated ~'3. Recent work has shown that ubiquitin conjugates are present in intracellular inclusions in degenerative diseases of the central nervous system 21, and o t h e r diseases. These structures include neurofibrillary tangles and senile plaques of A l z h e i m e r ' s disease 2~ 32 Lewy bodies of Parkinson's disease 21'23, neurofibrillary tangles of progressive s u p r a n u c l e a r palsy ~2">, and Mallory bodies of alcoholic liver disease, cytoplasmic bodies of cytoplasmic body inclusion m y o p a t h y , and Rosenthal fibers in astrocytes 2j. All of these inclusions contain i n t e r m e d i a t e filament proteins.

Corre,v~ondence: P.G. Galloway, Department of Pathology, Children's Hospital Medical Center of Akron, 281 Locust Street,

Akron, OH 44308, U.S.A. 00(16-8993/89/$03.50 © 1989 Elscvier Science Publishers B.V. (Biomedical Division)

344 However, the precise nature of the ubiquitinated proteins in these inclusions remains undetermined. Recent data also suggest that the ubiquitin system is present in human and animal neurons and plays a protective role in neurites under stress conditions 24" 2_~. Ubiquitin also functions in the cellular responses to other stresses including nutritional deprivation, viral intection, and to deleterious stimuli in general 21. Astrocytomas 4"6"73~'4~'43, as well as normal and reactive astrocytes ~'~'42"46, contain the intermediate filament cytoskeletal protein glial fibrillary acidic protein (GFAP), as demonstrated by immunocytochemistry. Some immunocytochemical studies showed that the more malignant, less differentiated astrocytomas are less likely to immunostain for GFAP than were the more differentiated tumors, and that the distribution of GFAP positivity within cells varied according to degree of differentiation. Another interesting aspect of astrocytomas is that when they are established as permanent cell lines in vitro 47, the expression of GFAP is eventually lost. However, in serial passages of glioblastomas in nude mice GFAP expression becomes reduced but never disappears ~9'22. The biological significance of these findings is unknown. Thus, although it has been clearly shown that GFAP is present in astrocytomas in vivo, the precise relationship between GFAP and biological differentiation and evolution of astrocytomas at the cellular level is complex. Virtually all the previous investigative work on the biochemical composition of astrocytomas involves various aspects of alteration of intermediate filament protein composition, specifically GFAP and vimentin 1~'33"34"36"45.By using light microscopic immunocytochemistry and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in this study, we investigated whether ubiquitin is specifically associated with GFAP in astrocytomas. We also investigated differences in ubiquitin epitopes in normal and reactive astrocytes versus neoplastic astrocytes. We show that antibodies directed against ubiquitin strongly stain astrocytoma cells in vitro and vivo, and that it co-distributes with GFAR We discuss our results in relation to current understanding of the role of ubiquitin in cellular functions.

MATERIALS AND METHODS A total of 27 tumor specimens were available for examination including 1 low-grade hemispheric astrocytoma, 5 anaplastic astrocytomas (including one gemistocytic astrocytoma), 15 glioblastomas multiforme, 4 cerebellar astrocytomas, 1 oligoastrocytoma and 1 optic nerve glioma. Controls consisted of normal brain or brain with reactive astrocytes removed at surgery or autopsy. Tissues were obtained immediately after surgical removal (in the case of biopsies), or less than 5 h post-mortem. O

Conventional light microscopy Samples were fixed in 10% buffered formalin and embedded in paraffin. Conventional sections were stained with hematoxylin and eosin. Tumors were classified according to the World Health Organization criteria 4~.

lmmunochemistry Paraffin sections of tissue, and cultured cells, were immunostained by the peroxidase-antiperoxidase (PAP) method of Sternberger 4°, with modifications as previously described 1~. An affinity-purified polyclonal GFAP antibody was used for the PAP method on paraffin-embedded tissue and cultured cells. Double labelling immunofluorescence was performed by incubating frozen tissue sections fixed in 95% alcohol, with primary antibodies (anti-ubiquitin and mouse monoclonal anti-GFAP [ICN Laboratories]) diluted in phosphate-buffered saline (PBS) pH 7.6. They were incubated with swine anti-rabbit IgG conjugated to rhodamine and swine anti-mouse IgG conjugated to fluorescein. Incubation was 20 min at room temperature (RT) and slides were rinsed 3 times in PBS between antibodies. Controls consisted of ubiquitin antibody absorbed with ubiquitin (Sigma) overnight at 4 °C, then centrifuged at 10,000 g for 30 rain at 4 °C. The supernatant was used for immunocytochemistry.

Cell culture Tumors for cell culture were cleaned of meninges and vessels, minced in Hank's balanced salt solution (HBAA) (Gibco), incubated in 0.01% collagenase Type II (Sigma) in HBSS at 37 °C for 5 min. Specimens were plated in 35-ml tissue culture flasks

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Fig. 1. Light microscopy of ubiquitin immunoreactivity. A: glioblastoma multiforme section stained with affinity purified ubiquitin antibody. B: serial section of A, stained with polyclonal GFAP antibody, x237, PAP with hematoxylin counterstain. C, D: cultured glioblastoma cells immunostained with anti-GFAP (C) and ubiquitin (D) antibodies by-PAP method. C: brightfield x422; D: Nomarski ×422.

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Fig. 2. Glioblastoma multiforme frozen section stained with A affinity-purified ubiquitin antibody and swine anti-rabbit immunoglubolin conjugated to rhodamine and B simultaneously with monoclonal GFAP antibody and swine anti-mouse immunoglobulin conjugated to fluorescein (x714). Note that ubiquitin and GFAP co-distribute in cell processes and cytoplasm.

(Falcon), and gassed in 5% CO 2 in balanced air, and incubated at 37 °C. Chamber slides and coverslips were plated with specimen at original plating and at subsequent passage levels, and fixed in 95% alcohol for subsequent immunostaining. One glioblastoma multiforme-estabtished cell line was purchased (American Type Culture Collection).

paper dotted with ubiquitin and immunostained with anti-ubiquitin, and a blot of human white matter immunostained with anti-GFAP, were stained. These results show that the polyclonal GFAP antibody was not cross-reacting with ubiquitin (i.e. that the immunostaining on tissue sections was not due to cross-reactivity of anti-GFAP with ubiquitin).

Characterization of antibody

Biochemical analysis

The characterization of the afffinity-purified polyclonal GFAP and ubiquitin antibodies has been previously reported 15,44. To further characterize the polyclonal GFAP antibody, ubiquitin (Sigma) (1 mg/ml) was dotted onto nitrocellulose paper and immunostained with anti-GFAP at concentrations from 1:10 through 1:5000. Immunostaining was not observed. In the same experiment, nitrocellulose

For biochemical analysis, control and tumor tissue was homogenized in a Dounce homogenizer (Kontes Glass Co.) in 0.1% SDS in phosphate-buffered saline (PBS) pH 7.6 and phenyl-methyl-sulfanyl fluoride (PMSF). Protein concentration was determined in aliquot samples by the method of Bradford z. Specimens were then suspended in sample buffer consisting of 2% SDS and 10% fl-

347 mercaptoethanol in 50 mM Tris-HC1, pH 6.8 and subjected to SDS-PAGE 2° using discontinuous 10% gels. Samples of tumors, controls, and molecular weight standards (Sigma Chemicals, St. Louis, Missouri) were electrophoresed. A section of gel including standards and specimen was stained with Coomassie blue for visualization of proteins. The proteins in the unstained gel were electrophoretically transferred to nitrocellulose paper (Schleicher and Scheull) as previously described 1~ or at RT at 1.7 Amp for 2 h. Immunobiotting was done 29. Briefly, the blots were incubated in 10% normal goat serum (NGS) in Tris-buffered saline (TBS) (37 °C for 1 h). Strips to be immunostained with ubiquitin antibody were boiled in water for 30 rain prior to immunostaining. The strips were incubated with the primary antibody (RT for 2 h), then rinsed with 0.05% Tween 20 in TBS at RT~ then with secondary antibody-peroxidase conjugate (1 : 1000 in 1% NGSTBS) (Cappel Laboratories) for 1 h at RT. They were rinsed in 0.05% Tween 20 in TBS for 1 h at RT then in 0.05 M Tris-HC1 at pH 7.6 twice for 10 rain each, followed by development in diaminobenzidine (0.75 mg/ml in 0.05 M Tris-HCl at pH 7.6) for less than 5 min. RESULTS

Light microscopy Although there was a diffuse background staining in tissue sections of both gray and white matter with the ubiquitin antibody, specific staining of normal astrocytes was not seen. Astrocytes were not specifically stained in regions of the brain where they are normally present, nor were cells with multiple processes characteristic of the morphology of fibrous or protoplasmic astrocytes immunostained in white or gray matter. There was no specific staining of the subependymal glial network, perivascular glial feet or the radial fibers of the Bergmann glia in the cerebellum. In cerebral neocortex, specific immunostaining was not seen in the external glial membrane or in astrocytes in the molecular layer. Immuno-

Fig. 3. Section of anaplastic astrocytoma stained with affinitypurified ubiquitin antibody (A) and after absorption with ubiquitin (B). Note immunostaining is removed by the absorption (x237, PAP with hematoxylin counterstain).

348

1

1'

1"

2

2'

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Fig. 4. Immunoblotting of human brain and astrocytoma preparations. A: lane on left is Coomassie blue-stained gel of cerebella~ astrocytoma; lane on right is blot-immunostained with ubiquitin antibody. Arrowhead points to band of about 60-kDa consistent with ubiquitinated GFAP. Molecular weight markers are as shown. B: lane 1 Coomassie-stained gel of glioblastoma multiforme. Blots of gel in Lane 1 immunostained with GFAP (Lane 1") and ubiquitin (Lane 1") antibodies. Arrowheads point to GFAP and ubiquitin bands respectively. Lane 2: Coomassie-stained gel of brain with reactive astrocytes. Lane 2': blot of gel immunostained with ubiquitin antibody. Arrowhead points to faint ubiquitin band. Molecular weight standards are as marked.

staining of reactive astrocytes with the ubiquitin antibody was not more prominent than background. In contrast to normal and reactive astrocytes, immunostaining of neoplastic astrocytes was intense (Fig. 1). Immunocytochemistry on sections of paraffin-embedded astrocytomas showed the presence of ubiquitin and GFAP in the cytoplasm and processes of tumor cells (Fig. 1A,B). This pattern was also seen in cultured astrocytoma cells (Fig. 1C,D). Double labelling immunofluorescence on frozen sections of tumors again showed staining in the same distribution (Figs. 1 and 2A,B). Immunostaining was

seen in all tumors regardless of degree of diferentiation or site in the neuraxis, and was more intense in the perikaryon than in cell processes (Figs. 1A-D and 2A,B). Staining by the ubiquitin antibody was removed by absorption with ubiquitin (Fig. 3). Rosenthal fibers in cerebellar astrocytomas showed strong staining of the periphery of the Rosenthal fibers with no staining of the central portion.

Biochemical analysis Representative examples are shown in Fig. 4. Immunostaining of blots of all normals, brain with

349 reactive astrocytes and astrocytomas showed a band of about 51 kDa consistent with GFAP. Immunostaining of blots from the same specimens with the ubiquitin antibody in most cases showed a low molecular weight band of about 16 kDa, consistent with ubiquitin dimers. Some astrocytomas showed a band of about 60 kDA consistent with ubiquitinated GFAP: others did not. DISCUSSION Although light microscopic evidence suggested that GFAP in astrocytomas was consistently ubiquitinated, this was not confirmed by the blotting experiments. Were GFAP ubiquitinated, a band corresponding to about a 60 kDa protein should have been shown in all tumor blots stained with the ubiquitin antibody. This finding was only seen in some tumors but not consistently. Thus, the identity of the ubiquitinated protein(s) in astrocytomas was not firmly established. Two explantions are possible: (l) GFAP is not necessarily the ubiquitinated protein in neoplastic astrocytes or (2) a conformational change in the molecule after SDS-PAGE affects the immunoblotting experiments. Certain epitopes may be detected by immunocytochemical methods only when associated with a particular structure or a certain conformation. Ubiquitin conjugation leads to conformational alterations expressed as distinct epitopes 3°. Other cell structures, e.g. microtubule components, are also only demonstrated by immunochemistry when they are assembled 2v. If GFAP is ubiquitinated, the ubiquitin antibody may only recognize the molecule in situ but not after denaturation. However, these data do suggest that the simultaneous presence of ubiquitin epitopes and intermediate filament proteins in various cellular structures, as demonstrated by light microscopic immunocytochemistry alone, does not prove that the intermediate filament proteins are ubiquitinated. Ubiquitin may be incorporated independently into those structures, or it may be attached to other as yet unidentified proteins. Ubiquitin, a 76-amino acid highly conserved protein, is thought to play a regulatory role in eukaryotic cells by forming conjugates to acceptor proteins '~m17"1~'~'39. Some of these conjugates are believed to be obligatory intermediates in the ATP-

dependent non-lysosomal proteolytic system that is particularly active in the degradation of damaged or abnormal proteins ~'2s. Thus, one of the roles of ubiquitin in neoplastic astrocytes may be to prevent or limit cell damage caused by the presence of altered cellular proteins. One approach in the study of astrocytomas is the elucidation of the processes responsible for the transformation of components normally present in astrocytes, into structures unique for the neoplastic state. The fact that by light microscopy ubiquitin epitopes are present in the cytoplasm and processes of astrocytoma cells suggests that the mere presence of ubiquitin stable conjugates is unique to the neoplastic state. Of two possible conclusions regarding the presence of conjugated ubiquitin in altered astrocytes, two are possible: (1) the proteins in the cells are ubiquitinated as a cellular response to the disease state, or (2) ubiquitin plays a role in processing or reorganizing the cell leading to transformation. Both of these interpretations suggest a deficit in the ubiquitin-mediated proteolytic pathway, since the ubiquitin conjugates are stable. However, absence of the accumulation of ubiquitin in normal and reactive astrocytes (as demonstrated by immunostaining normal brain sections with antiubiquitin) suggests that alteration of the ubiquitinmediated protein degradation system in malignant transformation may be specific to that condition. However, until the ubiquitinated proteins in neoplastic astrocytes are precisely identified, this question remains open, as the ubiquitinated proteins may be related to different cellular structures and functions. Comprehending the functional sequelae of ubiquitination of proteins in astrocytomas requires further analysis. In astrocytomas, either internal or external forces may disturb the normal regulatory environments interfering with both the normal degradative and other undetermined regulatory roles of ubiquitin 31. One possibility is analagous to that postulated for the role of ubiquitin in neurofibrillary tangle formation in Alzheimer's disease neurons 3°. That is that in affected cells, in this case abnormal astrocytes, a disruption of the normal degradation process of cytoskeletal components develops, leading to accumulation of partially-proteolyzed, ubiquitinated proteins, The increase in stable ubiquitin

350 conjugates may be due to a saturation of the u b i q u i t i n - m e d i a t e d proteolytic system due to the presence of increased amounts of altered proteins 23, because the ubiquitinating and u b i q u i t i n - d e p e n d e n t proteolytic activities have been found to be m a i n t a i n e d 3'39. The ubiquitin system might become saturated by altered cytoskeletal proteins which would collect in cytoplasm and processes of cells. It could also be due to sustained cellular stress related to the disease processes z~. Identification of the ubiquitin acceptor proteins in pathologically altered astrocytes may p r o v i d e new knowledge into mechanisms of cellular reorganization in disease states, and elucidate new functions for the ubiquitin system in the p a t h o l o g y of the central nervous system. In s u m m a r y , we provide additional evidence that the ubiquitin system is operative in the central

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ACKNOWLEDGEMENTS The authors thank Mr. Vince Messina and Mr. R o b e r t Langlotz for assistance with medical photography, Ms. Vicki Kasmin and Mrs. R o s e D e k a for help in p r e p a r i n g the manuscript, and Dr. A r t h u r H a a s for the ubiquitin antibody. This work was s u p p o r t e d by C u y a h o g a C o u n t y Hospital F o u n d a tion G r a n t and Case Western R e s e r v e University Cancer C e n t e r G r a n t P30CA43703, to P . G . G .

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