Can permanent cell lines originate from low-grade astrocytomas: overgrowth of "glia-like" cells in pilocytic and fibrillary (diffuse) astrocytoma cultures.

OBJECTIVES: Due inter alia to wide-spread cell lines cross-contamination it is not clear, which kind of normal or tumoral tissue give rise to permanent cell lines. BACKROUND: Few permanent cell lines have been established from low-grade astrocytomas. However, recently some of these have been identified as being cross-contaminated with other cell lines. METHODS: Morphology, cell growth and GFAP immunophenotype of low-grade astrocytomas were examined on 9 pilocytic and 15 fibrillary (diffuse) tissue cultures. RESULTS: GFAP-positive process-bearing cells were present in all the cultures, mainly during the first days in vitro (DIV). In pilocytic cultures, cells with hairy (piloid) processes were present. GFAP-positive cells completely disappeared by passages 3 to 5 and all the cultures contained only GFAP-negative "glia-like" cells, which underwent cellular senescence within passages 8 to 15. CONCLUSION: Key differences in the morphology and GFAP expression between the neoplastic astrocytes and normal "glia-like" cells allow the observation of perceptibly more rapid growth of normal cells in astrocytoma cultures. We caution that cultures prepared from macroscopically tumoral brain tissue may contain rapidly proliferating normal cells. Based on this and our previous studies in relation to the high percentage of cross-contaminated cell lines, we conclude that cells in low-grade astrocytoma cultures lack the capacity for spontaneous immortalization (Fig. 14, Ref. 15). Text in PDF www.elis.sk Keywords: pilocytic astrocytoma, fibrillary astrocytoma, "glia-like" cells, glioma cell lines, GFAP.

Short-term glioblastoma cultures may contain normal "glia-like" cells.

OBJECTIVES: Currently used glioblastoma cultures have many disadvantages and are being replaced by short-term cultures. However, these may include normal brain cells. BACKGROUND: A comparative model of normal and glioma cultures is lacking. A significant contributory factor is because cultures from adult human brain contain small amounts of cells with glial phenotypes. The predominant population of flat or spindle shaped cells does not express glial markers and are often termed as "glia-like". METHODS: Cryopreserved glioblastoma cultures from 28 bioptic samples were examined by immunofluorescence using antibodies to intermediate filaments (IF): glial fibrillary acidic protein (GFAP), cytokeratins (CK), nestin (Nes), vimentin (Vim) and neurofilaments (NF). RESULTS: In short-term glioblastoma cultures GFAP-positive cells occured at higher percentages in 3/28 cultures and in lower percentages in further 5 cultures. Subpopulation of nestin positive cells were observed in all cultures and CK-positive cells were found in 25/28 cultures. All cells in all cultures were positively stained only for vimentin and negatively for NF. Cells grew slowly in 5 cultures which showed early proliferation arrest between passages 7 to 8. A further 23 cultures showed growth arrest by passages 10 to 15. CONCLUSION: The presence of normal cells in short-term glioblastoma cultures may be caused by the infiltrative growth of these tumors. Our comparative analysis of morphological, growth and cytoskeletal properties revealed similarities between glioblastoma and normal brain cultures. In this study, the majority (28/30) of short-term glioblastoma showed limited life spans, similar to normal cells lacking spontaneous immortalization. The use of short-term glioblastoma cultures has two main problematic areas: cultures may contain a major subpopulation of normal "glia-like" cells; or they may contain the inital phases of spontaneously immortalized glioblastoma cells bearing properties of permanent cell lines (Tab. 1, Fig. 2, Ref. 19).

High level EGFR amplification in a newly established glioblastoma cell line 170-MG-BA.

Glioblastoma multiforme is a highly invasive and incurable primary brain tumor. The most frequent genetic alteration therein is amplification of the epidermal growth factor receptor (EGFR) gene, the target of current clinical trials. However, EGFR amplification is poorly represented in glioblastoma cell lines. From the 30 cultures attempted herein, we were able to establish two glioblastoma permanent cell lines. The remaining cultures showed limited life span and underwent senescence between passage numbers (PN) 8 to 15. Our newly established glioblastoma cell lines, designated 170-MG-BA and 538-MG-BA, both originated between PN 3 and 5 when areas of smaller, more rapidly proliferating cells appeared. Both cell lines showed similar rates of growth, moderate morphological differences, cytoskeletal heterogeneity and multiple chromosome rearrangements. Analysis by molecular cytogenetics and comparative genomic hybridization (aCGH) revealed two copies of a stable marker chromosome in 170-MG-BA cells effecting focal amplification at 7q11 of the EGFR locus. Comparative RqPCR analysis confirmed that EGFR was uniquely highly expressed in 170-MG-BA cells. Combined targeted expression analysis and aCGH data excluded the recurrent EGFRvIII activating mutation. In contrast, EGFR expression in 538-MG-BA cells which lacked genomic EGFR amplification was not raised. Immunofluorescent staining showed high EGFR protein expression only in the 170-MG-BA cells. Cytogenetic, genomic and transcriptional analyses then confirmed high-level genomic amplification and transcriptional upregulation of wild type EGFR in 170-MG-BA; the first conventional cell line model for investigating the biology and targeted therapy of this key alteration in glioblastoma. Both cell lines are freely available from the DSMZ cell repository.

The vascular patterns of the posterior part of the circulus arteriosus cerebri (Willisi).

BACKGROUND: Various authors defined three patterns of the posterior part of the circulus arteriosus cerebri Willisi (CW) according to the diameter of the posterior communicating artery (PCoA) and the precommunicating segment of the posterior cerebral artery (P1). In the adult pattern, the P1 has a diameter larger than the non-hypoplastic PCoA. In the transitional pattern, the diameter of the PCoA is equal to that of the P1. In the fetal pattern, the diameter of the P1 is smaller than the diameter of the PCoA. The study was aimed to evaluate the configurations and calibers of the posterior part of the CW. METHODS: The work was conducted on 185 adult post-mortem brains. The CW and its branches were photographed by a digital camera. We used the software Image J to evaluate and process the gained images. RESULTS: The fetal pattern was found unilaterally in 8.37 %, and bilaterally in 4.86 %. The transitional pattern was observed unilaterally in 6.47 %, and bilaterally in 1 %. The prevalence of the unilateral and bilateral adult patterns was equal (21.62 % for each configuration). The hypoplastic PCoA was found unilaterally in 17.57 %, and bilaterally in 16.76 %. CONCLUSION: Various factors including genetic and environmental may affect the development of the cerebral vessels and their dimensions. The distinguishing of the vascular dimensions in vivo can help in the expectation and may be the avoidance of possible cerebrovascular disturbances in the future. Correlation and interdisciplinary cooperation of the studies dealing with morphology, radiology, and hemodynamics of the cerebral vessels are becoming an urgent need. The assumed results of this cooperation can be used in tabulating the calibers of the cerebral vessels and determining the threshold dimensions under which failure of hemodynamics and collateral function may appear (Tab. 2, Fig. 5, Ref. 28).

Subserosal localization of myenteric ganglia in normal human appendix: immunostaining with neuronal and glial markers.

OBJECTIVES: Visualization of unexpected distribution of myenteric ganglia in normal human appendiceal wall by immunofluorescence. BACKGROUND: The myenteric plexus is located between the longitudinal and circular muscle layers of the GIT. However, recently the irregular distribution of myenteric ganglia was revealed in human appendix. METHODS: The cryosections prepared from normal human appendices were examined by immunofluorescence methods using antibodies to neurofilaments (NF) and glial fibrillary acidic protein (GFAP). RESULTS: Indirect immunofluorescence revealed the positive staining of myenteric ganglia with both neuronal and glial marker antibodies. Double labeling for NF/GFAP staining showed close assotiation between glia and neurons inside ganglia. GFAP-positive cells were often observed as the cells surrounding myenteric ganglia. The staining confirmed the irregular distribution of myenteric ganglia in human appendiceal wall and revealed the small ganglia in the subserosal area. CONCLUSION: Our results showed that localization of myenteric ganglia in human appendix differs from other parts of GIT. GFAP immunostaining is available for visualization of smaller myenteric ganglia located mainly in the subserosal area. Our studies may find application in current HIV research focused on enteric neuropathogenesis and in diagnostic laparoscopy for chronic abdominal pain: to detect the irritation of subserosal ganglia (Fig. 2, Ref. 26).

Biological aspects of appendix pathology: irregular distribution of myenteric ganglia in human appendiceal wall.

OBJECTIVES: Although appendicitis is a common disease, basic questions about risk factors and its etiology remain unexplained. BACKGROUND: An obstruction of the appendix lumen is usually considered to be the main cause of acute appendicitis. However, more studies are currently dealing with neuroimmune appendicitis. METHODS: We studied samples of human appendices with the histological diagnosis of chronic appendicitis. Fixed cryosections of appendiceal walls were examined by immunofluorescence methods using neuronal anti-neurofilament antibody markers and beta III tubulin. RESULTS: The immunostaining revealed an irregular distribution of myenteric ganglia in inflamed appendiceal walls and unexpected groups of large ganglia unequally distributed in the subserosal area. The comparative analysis of normal and inflamed appendix samples showed differences in the occurrence of myenteric ganglia in the subserosal area. They appeared more frequently on cryosections prepared from the inflamed appendiceal wall. CONCLUSION: We propose that the high variability and irregular location of myenteric ganglia in the appendiceal wall are due to an alteration in the motility which results in flaccid appendix emptying. In addition, superficially located myenteric ganglia are exposed to abdominal irritation and may explain the chronic abdominal pain which is often considered to be a sign of chronic appendicitis (Fig. 2, Ref. 23).

Unexpected presence of cytokeratins in human "glia-like" cells.

OBJECTIVES: To demonstrate the unexpected features of the predominant cell population in adult human brain tissue cultures usually termed "glia-like" cells. BACKGROUND: Cytokeratins (CK) are intermediate filaments (IF) specific for normal and neoplastic epithelial cell differentiation. METHODS: We examined adult human brain tissue cultures and cryosections prepared from ten biopsies. Immunofluorescence staining with monoclonal antibodies against IF proteins: anti-pan CK, glial fibrillary acidic protein (GFAP) and vimentin was performed on primary and secondary cultures up to passage 10. RESULTS: In primary cultures we detected only small numbers of immunocytochemically distinct astrocytes, oligodendrocytes and microglial cells. "Glia-like" cells were negatively stained with specific glial marker antibodies. They were positively stained with pan-CK antibodies in 8/10 cultures where 0.1 % to 70 % CK-positive cells were present in primary as well as in secondary cultures. Comparative immunofluorescence IF staining for CK, GFAP and vimentin showed differences in the cytoplasmatic distribution of IF fibres, numbers of positive cells and intensities of staining. Cryosections from brain biopsies stained negatively with pan-CK antibodies. CONCLUSION: These findings demonstrate the presence of CK in adult human brain cultures which is not caused by cross-reactivity of IF antibodies. Based on these results we propose that unexpected CK expression in human "glia-like" cells is due to cell dedifferentiation under culture conditions (Tab. 1, Fig. 2, Ref. 24).

Does incorporation of gene for green fluorescent protein in BP6 fibrosarcoma tumor cells depress their intraperitoneal growth in rats? (In honour of Nobel Prize laureates 2008--Osamu Shimomura, Martin Chalfie, Roger Y. Tsien).

This manuscript was in honour of Nobel Prize in chemistry "for the discovery and development of the green fluorescent protein, GFP" to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien, simultaneously a brief information about experience with GFP in experimental tumorigenesis used this study is also presented. The experimental data have showed that BP6 cells incorporated with GFP gene have had smaller ability to induce both experimental intraperitoneal and subcutaneous tumor process. It was anticipated that incorporation of GFP gene might change physiological properties of cytoskeleton and worsen adhesive characteristics of tumor cells. It was also supposed that aftertime GFP will enable to monitor proliferation of cells not only within experimental work, but also in human medicine. GFP could help (supposedly) as reporter of proliferation, but also can serve as "target" for guide of tumorigenesis inhibiting substances. These ideas which are consequences of our experiments we append as congratulation to Nobel Prize in chemistry of the 2008 (Fig. 2, Ref. 44). Full Text (Free, PDF) www.bmj.sk.

Effects of a novel synthetic retinoid on malignant glioma in vitro: inhibition of cell proliferation, induction of apoptosis and differentiation.

Among six synthetic retinoids tested, the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) was highly efficient in inducing growth inhibition of 8MG-BA and GL-15 human glioblastoma cell lines, with growth arrest at the S phase of the cell cycle. CD 437 also induced apoptosis in these cells, with 8MG-BA being the most sensitive. In these cells, induction of apoptosis by CD437 has been related to the downregulation of Bcl-2 expression and to CPP32 activation, but not to p53 expression. The remaining non-apoptotic cells presented a morphological pattern of astroglial differentiation with overexpression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The mechanism of action of CD437, originally developed as a RARgamma agonist, is not yet elucidated. However, our results suggest that it acts through an increase of the expression of retinoid-inducible genes, such as RARbeta2 and/or RARalpha2.

Differential expression of laminin and fibronectin and of their related metalloproteinases in human glioma cell lines: relation to invasion.

In the present work, we analyzed the expression of two major components of the extracellular matrix (ECM), laminin and fibronectin and of two related matrix-metalloproteinases, MMP-2 and MMP-9, in three human glioma cell lines (8 MG, 42 Mg and GL-15) in relation with their differential invasive properties. Immunocytochemistry and Western-blots assays indicated the presence of a 200 kDa laminin, similarly expressed in the three cell lines but undetectable in their ECM. In the opposite, a 230 kDa fibronectin, detected in the three cell lines was differently expressed and only observed in the ECM of the less invasive 8 and 42 MG cells. MMP-2 mRNA analyzed by Northern blots and proMMP-2, evaluated by zymography, were found in the three cell lines but were both ten times higher in the most invasive GL-15 cells. In addition, the active form of MMP-2 was only found in the GL-15 cells. In the opposite, the expression of specific tissular inhibitor (TIMP)-2, an endogenous MMP-2 inhibitor, was restricted to the less invasive cells. MMP-9 activity was detected only in the 8 and 42 MG cells and may not be directly involved in invasion. Taken together, these results indicate that a high MMP-2/TIMP-2 ratio may be responsible for the absence of extracellular fibronectin, underlining the participation of tumour cells in the proteolytic degradation of the ECM. An unbalanced MMP-2/TIMP-2 ratio in the micro-environment of malignant cells may contribute to their invasive properties.

Establishment, morphological, growth and cytoskeletal properties of 135-BCA carcinoma cell line derived from lung brain metastasis.

Many cell lines have been established from lung cancer but carcinoma cell lines derived from brain metastases occur rarely. The carcinoma cells growth relatively slowly in comparison with brain cells which often overgrow the tumor cells in early passages. The origin of these rapidly dividing brain cells in carcinoma cultures is discussed with respect to the previous studies on adult human brain tissue cultures. It was found that the majority of cells in adult human brain cultures derived from brain biopsies of patients with non-cancer diseases do not express glial markers. Based on the previous studies we suggest that they are glial precursor cells. The high proliferative capacity and non-glial phenotype of these brain cells may lead to the suggestion that they are of cancer origin. In this study the establishment and characterization of a new carcinoma cell line 135-BCA is described. The tissue cultures were derived from brain metastasis of lung large cell carcinoma. The cell line is specific by the epithelial cell morphology and evident cytokeratins expression during the whole subcultivation. All tumor cells were strongly immunoreactive for vimentin and negative stained for glial fibrillary acidic protein (GFAP). The new cell line may prove of value in biological and therapeutic studies of lung cancer. In addition, the further comparative analysis may reveal the environmental influence of brain tissue on carcinoma cells.

Heterogeneity of keratin intermediate filaments expression in human glioma cell lines.

Keratin intermediate filaments (Ifs) are specific for epithelial cell differentiation. This study demonstrates the presence of keratin in two recently established human glioblastoma cell lines 8-MG-BA and 42-MG-BA. Immunofluorescence staining was performed on cells within passage 230 to 235 using monoclonal pan-cytokeratin antibodies. The cells were analyzed during several DIV at different cell density. Keratin-positive stained cells reached 5 to 7% in 8-MG-BA and less than 0.1% in 42-MG-BA cell line. The presence of keratin-positive cells was independent on cell density and days in vitro. Keratin-positive cells appeared unevenly distributed in both cell lines. They were observed as single or areas of keratin-positive cells. The morphological features of keratin-positive and keratin-negative cells were similar. The results are discussed with respect to previous studies on glial fibrillary acidic protein (GFAP) and vimentin to show the heterogeneity of IFs expression in glioma cell lines.

Characterization of two new permanent glioma cell lines 8-MG-BA and 42-MG-BA.

The establishment and characterization of two permanent glioma cell lines (8-MG-BA and 42-MG-BA) are described. Both cell lines were derived from the human glioblastoma multiforme. Analyzed cells were within the passage 200 to 220. The cells in both cultures showed similar morphology. In majority they consisted from flat polygonal cells. Growth kinetic studies demonstrated a population doubling time of 20 to 24 h in cell line 8-MG-BA and 48 to 54 h in cell line 42-MG-BA. The cell lines showed different hyperdiploid karyotypes. The immunofluorescence staining was performed for glial fibrillary acidic protein (GFAP) and vimentin. In the culture 8-MG-BA only a small amount of cells showed the GFAP-positive staining. At confluent 42-MG-BA culture the GFAP-positive cells reached 50 to 70% of all cells. Vimentin was found in all glioma cells in both cultures.