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Biomedical subjects

R Bjerkvig

Publications and source records attributed to R Bjerkvig.

At least 73 records · Page 4Linked to original sources
PubMed1991

Anti-GM2 monoclonal antibodies induce necrosis in GM2-rich cultures of a human glioma cell line.

The effects of four anti-GM2 monoclonal antibodies (DMAb-1, DMAb-2, DMAb-3, and DMAb-5) were studied on spheroid cultures from a human glioma cell line (D-54 MG) that is known to express high levels of GM2. The spheroids developed central necrosis 48 h after antibody exposures at concentrations greater than 6 micrograms/ml. No necrosis was found with antibodies that had been absorbed with GM2 prior to exposure or with unrelated cytotoxic antibodies. Immunohistochemistry showed that the necrosis started shortly after the antibodies were evenly distributed throughout the spheroids. Light and transmission electron microscopy revealed that a small portion of the cells, mainly in the periphery of the spheroids, was unaffected by antibody exposure. New monolayer cultures established from antibody-treated cells expressed a 50% lower GM2 content as shown by flow cytometry and determination of ganglioside content throughout at least 12 passages. Thus, the GM2-rich D-54 MG cell line has subpopulations of cells with lower GM2 content. Spheroids obtained from this subpopulation developed only minor necrosis after antibody treatment. These results show that GM2 antibodies cause severe necrosis of GM2-containing glioma cells in vitro, but the effect depends on the concentration of antigen, and a threshold number of GM2 molecules is required.

Antibodies, Monoclonal↗
PubMed1991

Deletion-mutant epidermal growth factor receptor in human gliomas: effects of type II mutation on receptor function.

Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.

Amino Acid Sequence↗
PubMed1991

Effect of alkyl-lysophospholipid on glioblastoma cell invasion into fetal rat brain tissue in vitro.

The antitumor effect of alkyl-lysophospholipid (ALP) was studied on a continuous glioma cell line (GaMg) as well as on tumor spheroids obtained from three different primary brain tumor biopsies. GaMg monolayer growth was reduced by 50% after treatment with 30 microM ALP; cells accumulated in the G2M phase of the cell cycle as determined by flow-cytometric analyses. Tumor spheroid growth was reduced by 25 and 44% during treatment with 10 and 30 microM ALP, respectively. These drug concentrations also caused a severe destruction of spheroids. No effect on growth or morphology was seen in spheroids treated with 0.1 and 1.0 microM ALP. ALP caused a dose-dependent inhibition of invasion by GaMg tumor spheroids into brain aggregates. After 168 h of 1.0 microM ALP treatment, the volume of the intact brain aggregate was 90% larger than that in the untreated co-cultures. To further investigate the efficacy of ALP as an anti-invasive drug, co-cultures were performed with specimens obtained from three primary brain tumors: a highly invasive glioblastoma multiforme, an anaplastic astrocytoma, and an astrocytoma. Treatment of spheroids from the most invasive tumor with ALP caused a 7-fold preservation of normal brain tissue relative to control co-cultures. Moreover, the sensitivity of primary glioma spheroids to the anti-invasive effect of ALP seemed to be associated with the aggressiveness of the tumor; spheroids from the more malignant specimen (glioblastoma multiforme) were more sensitive than those from the less aggressive tumors. The anti-invasive effect seen with nontoxic concentrations of ALP may prove valuable in the treatment of malignant gliomas.

Adult↗
PubMed1991

Characterization of NCAM expression and function in BT4C and BT4Cn glioma cells.

The neural cell adhesion molecule, NCAM, plays an important role in cell-cell adhesion. Therefore, we have studied NCAM expression in the glioma cell lines BT4C and BT4Cn. We demonstrate that the 2 cell lines differ in their metastatic ability; while BT4C cells have a very low capacity for producing experimental metastases, that of BT4Cn cells is high. In BT4C cells NCAM is synthesized as 4 polypeptides with Mr's of 190,000, 140,000, 115,000 and 97,000. The 140,000, 115,000 and 97,000 polypeptides are glycosylated and for the 140,000 and 115,000 polypeptides sulfatation is observed. Conversely, no NCAM protein synthesis is observed in BT4Cn cells, even though NCAM mRNA is expressed. Thus, development of an increased metastatic capacity is accompanied by the disappearance of NCAM protein expression in this model system. The functional importance of NCAM expression was studied by a cell-substratum binding assay in which the binding of BT4C and BT4Cn cells to NCAM immobilized to glass was assessed. We found that BT4C cells adhere specifically to NCAM, and that adhesion is inhibited by anti-NCAM Fab'-fragments, while no specific binding of BT4Cn cells to NCAM was observed. The BT4C and BT4Cn cell lines thus constitute an important new model system for the study of tumor invasion and metastasis and of the role of cell adhesion molecules in these processes.

Animals↗
PubMed1990

Isolation and characterization of a metalloproteinase secreted by rat glioma cells in serum-free culture.

The isolation of a metalloproteinase secreted by a rat glioma cell line (BT5C) in serum-free media is described. After affinity purification, the activity was present as a double band with Mr 86000 and 76000 both of which required CaCl2 for activity. The enzyme was able to degrade gelatin but not casein. It was unable to degrade native types I, III, IV and V collagens but their denatured counterparts were degraded. Using a radiolabel release assay the enzyme was inhibited by EDTA, 1:10 phenanthroline and TIMP confirming that it belongs to the family of metalloproteinases. Its activity was not affected by either serine or cysteine protease inhibitors. The proteinase was activated by APMA but was unaffected by trypsin treatment.

Animals↗
PubMed1990

Effect of epidermal growth factor on glioma cell growth, migration, and invasion in vitro.

Effects of epidermal growth factor (EGF) and an antibody (Ab-528) reactive against the binding site for EGF on human EGF receptors were studied on multicellular tumor spheroids obtained from three human glioma cell lines with high (D-37 MG), medium (D-247 MG), and low (D-263 MG) levels of EGF receptor expression. The D-247 MG and D-263 MG spheroids grew slowly or not at all in the absence of EGF, while in the presence of EGF they were growth stimulated. Tumor cell migration, as measured by the spread of cells from spheroids on a plastic substratum, was increased by the addition of EGF for all three cell lines. Stimulation of migration could be blocked by a subsequent addition of Ab-528 to the medium at a concentration of 50 micrograms/ml. Invasiveness of glioma cell spheroids into fetal rat brain aggregates was related to EGF receptor expression; the two lines with medium to high receptor expression (D-247 MG and D-37 MG) were invasive, while the line with low EGF receptor expression (D-263 MG) was noninvasive, as assessed by an in vitro coculture assay. In the D-247 MG cell line, morphometry revealed EGF-enhanced invasiveness of the tumor cells. The addition of the Ab-528 to EGF-treated cocultures reduced invasion in both D-247 MG and D-37 MG cell lines. Antibody Ab-528 alone did not affect glioma cell growth or migration but did inhibit invasiveness. The present study suggests that, in brain tumors with an increased number of normal-sized Mr 170,000 EGF receptors, EGF or an EGF-like ligand such as transforming growth factor-alpha may selectively facilitate expansive tumor growth and tumor cell invasion. This effect may in part be blocked or retarded by specific antibodies to the EGF receptor.

Cell Division↗
PubMed1990

Interaction between human brain tumour biopsies and fetal rat brain tissue in vitro.

The invasiveness of human intracranial tumours was studied in an organ culture system. Biopsies from six glioblastomas, four astrocytomas, two mixed gliomas, one ependymoma, four meningiomas and two carcinoma metastases were cut into fragments of 0.5 mm diameter, and placed in agar overlay tissue culture. The tumour specimens formed spheroids which were co-cultured with cell aggregates or fragments from fetal rat brain for up to 10 days in vitro. The invasiveness of the glioblastoma spheroids was characterised by a gradual destruction of normal brain tissue by tumour cells, followed by replacement of normal tissue by these cells. Co-cultures from two glioblastomas showed lesions in the normal brain tissue in areas removed from the tumour cells. Tumour spheroids from four glioblastomas totally destroyed the normal brain tissue without any change in the original tumour spheroid configuration. The low-grade gliomas were less invasive than the glioblastomas. The meningiomas and the metastases were non-invasive. This organ culture assay appeared to reflect the in situ invasive behaviour of the brain tumours examined. It is suggested that it may be used for evaluating the aggressiveness of individual brain tumours with the specific aim of correlating clinical data with the biological character of the tumour.

Adolescent↗
PubMed1990

A cerebral glioma model for experimental therapy and in vivo invasion studies in syngeneic BD IX rats.

An in vivo glioma model was developed in syngeneic BD IX rats. The BT4An tumor was derived from serial in vivo passages of the BT4A tumor, originally induced from transformed fetal rat brain cells after transplacental exposure to ethylnitrosourea. The cell line was characterized for the presence of neuroglial differentiation markers, chromosome content and cell cycle distribution as determined by flowcytometry. A standardized method for i.c. tumor induction was developed, and the tumors were investigated by light and electron microscopy and for evidence of blood-brain barrier disruption. Tumor cell ability for phagocytosis was studied, as this property may be important for the invasion pattern of the tumors. We conclude that the model seems suitable for both in vivo therapy and invasion studies. The tumor had 100% tumor take, yielded a predictable symptom-free life span after inoculation, had a characteristic histological picture of an aggressive glioma, and the blood-brain barrier within the tumor was in part disrupted. Compared to the parent cell line, there was loss of neuroglial differentiation markers, the chromosomal distribution was changed, and the ability for phagocytosis was practically lost.

Animals↗
PubMed1990

Multicellular tumor spheroids from human gliomas maintained in organ culture.

Tumor tissue from seven human gliomas was maintained in long-term agar overlay culture as multicellular organotypic spheroids. Light microscopic and ultrastructural observation of the spheroids displayed morphological features similar to those of the original tumor tissue in vivo; in this respect they were different from spheroids obtained from permanent cell lines. The spheroids contained preserved vessels, connective tissue, and macrophages, revealing a close resemblance to the conditions in the original tumor. Flow cytometric deoxyribonucleic acid measurements of cells from the tumor spheroids and from biopsy material obtained directly from the operation revealed the same ploidy and the same amount of proliferating cells in the spheroids as in the original tumor. Fluorescence microscopy using bromodeoxyuridine (BUdR) incorporation and anti-BUdR monoclonal antibody confirmed the proliferative potential of tumor cells in the spheroids. Diameter measurements showed that the size of the spheroids from two of the tumors increased over time while in three other cases it decreased. Spheroids from the remaining two tumors showed no change in size, even after 80 days in culture. These growth data and the relatively high number of proliferating cells, as measured by flow cytometry, indicate that the degree of cell proliferation and cell loss from the spheroids are closely linked, as is the case for tumors in vivo. The culture system presented provides a valuable alternative to propagation of human tumors in animals.

Biopsy↗
PubMed1989

Immunolocalization of extracellular matrix proteins during brain tumor invasion in BD IX rats.

The distribution of several native extracellular matrix proteins (type I, III, and IV collagens and fibronectin) using immunofluorescent localization is described for in two different malignant gliomas (BT4A and BT4An). In addition, antibodies against denatured forms of type I and III collagens were used to localize areas of active degradation within the tumors. We have shown that both tumors express the native connective tissue components studied, although the distribution of these components within and between the tumors was different. In addition, native type I and III collagens and fibronectin were overexpressed in the tumors compared to the normal brain. Morphometry on immunostained type IV collagen sections showed an increase in vascular elements in both tumors compared to normal brain tissue. The BT4A tumor, which by light microscopy showed a degradative mode of invasion, expressed denatured type I and III collagens at the tumor-brain border zone, suggesting that this tumor has collagenolytic activity. The present article suggests that the distribution and changes in extracellular matrix protein synthesis and degradation may play an important role in the progressive growth of brain tumors in vivo.

Animals↗
PubMed1989

Immunocytochemical characterization of extracellular matrix proteins expressed by cultured glioma cells.

The immunolocalization of type I, III and IV collagens and fibronectin in two rat glioma cell lines in vitro (BT4C and BT4Cn) is described. In addition, antibodies against denatured type I and III collagens were used to study breakdown products of native type I and III collagens. For the BT4C cells, the extracellular matrix expression in monolayer cultures and in multicellular tumor spheroids was compared. Type IV collagen was strongly expressed in BT4C tumor spheroids but was negative in the corresponding monolayer cultures. Denatured type I collagen was found both in monolayers and in spheroids of BT4C, suggesting either a rapid turnover (i.e., synthesis and immediate breakdown) of type I collagen or an altered collagen gene transcription. Both cell lines were negative for native type I and III and denatured type III collagen. Fibronectin was strongly expressed in both cell lines. Supporting the immunofluorescence data, the hydroxyproline content in the tumor spheroids was twice the amount found in monolayer cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting also verified the immunostaining experiments, showing that glioma spheroids and injected tumor cells have the potential for fibronectin and collagen production, given the appropriate growth conditions.

Animals↗
PubMed1988

Whipple's disease. Demonstration of a persisting monocyte and macrophage dysfunction.

A patient with Whipple's disease has been followed up for 4 years. Primary involvement was limited to the small intestines, and accumulation of periodic acid-Schiff-positive material, containing typical more or less intact bacillary bodies, was demonstrated within macrophages of affected tissue. After initial oxytetracycline treatment and clinical remission, the patient relapsed, with multiorgan affections. The antibiotic regimen was changed to chloramphenicol, followed by continuous trimethoprim-sulfamethoxazole. Flow cytometric studies showed persisting impairment of monocyte and macrophage intracellular degradation of bacteria during all the 4 years tested. After relapse, reduced activity of several brush border enzymes was demonstrated in distal duodenal biopsy specimens. After 17 months of continuous trimethoprim-sulfamethoxazole therapy complete clinical remission, regression of histopathologic abnormalities, and restoration of duodenal enzyme activities had occurred. The results demonstrate a persisting dysfunction of mononuclear phagocytes from a patient with Whipple's disease, suggesting a primary abnormality of cell-mediated immunity which may promote the susceptibility to the causative bacillus.

Adult↗
PubMed1987

Biological and biochemical characterization of cell lines derived from initiation-promotion transformed C3H/10T1/2 cells.

A two-stage transformation protocol was used to chemically transform the mouse embryo fibroblasts, C3H/10T1/2 Cl 8. To initiate the cells 0.37 microM 20-methylcholanthrene was used and 0.17 microM of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate was employed to complete the transformation process. Six weeks later transformed foci were identified and isolated by the ring-cloning technique. Altogether eight different foci were trypsinized resulting in a total of 12 morphologically transformed subclones. Three of these clones, designated TPA 41, TPA 42 and TPA 482, have been characterized in detail. Their growth morphologies were different. The TPA 482 cells grew in a criss-cross pattern with piled up foci, thus showing a characteristic type III morphology. The TPA 482 clone did not show cell-density growth inhibition and grew in soft agar. The TPA 41 and TPA 42 clones exhibited cell-density growth inhibition, grew as monolayers and formed only few colonies in soft agar. Late passages of the TPA 42 clone acquired growth characteristics similar to TPA 482. The C3H/10T1/2 Cl 8 and the TPA 41 cells were not tumorigenic when transplanted into syngeneic mice. TPA 482 cells were strongly tumorigenic, producing tumors in 6/6 mice in 21 days. The TPA 42 cells were also tumorigenic, the first tumors appearing after 4 weeks; all animals injected with TPA 42 cells had tumors after 8 weeks. All tumors observed appeared to be fibrosarcomas. Flow cytometric analysis indicated differences in DNA distributions between tumor cells grown in vitro and the tumors in vivo. Two-dimensional gel analysis of the total cellular and the nuclear proteins showed an increase in the TPA 42 and TPA 482 cells of an acidic 48,000 and a basic 83,000 mol. wt polypeptides, and a decrease of a neutral polypeptide of mol. wt 46,000, located in the nucleus of TPA 482 cells.

Animals↗
PubMed1987

Phagocytic capacity of normal and malignant rat glial cells in culture.

The phagocytic capacity of 4 continuous rat glioma cell lines (BT2C, BT4Cn, BT5c, and 9L) and normal BD IX fetal rat glial cells in culture has been studied. This was done by flow cytometric measurements of single cells from monolayer cultures having ingested fluorescent bacteria, zymosan particles, red blood cells, or fragments of normal glial cells. In addition, phagocytosis was studied in a three-dimensional culture system. The BT4Cn, BT5C, and 9L cell lines were tumorigenic and invasive both in vivo and in organ culture in vitro. In contrast, BT2C has shown variable tumorigenicity and does not seem to be invasive. The phagocytic capacity of the cell lines was compared to their destructive properties during invasion. Depending on the particle type, 30-40% of the normal glial cells were phagocytic. The fractions of phagocytic glioma cells were dependent on the particle type and the prey load. Of the invasive cell lines, BT5C showed high phagocytic activity both in monolayer and three-dimensional cultures. Two of the invasive cell lines (BT5C and 9L) had about the same fraction of phagocytic cells as normal glial cells. These 2 cell lines showed highly destructive growth during invasion. In contrast, the third invasive cell line (BT4Cn) had almost no phagocytic cells. The BT4Cn cells showed single-cell invasion with little destruction of target tissue. The noninvasive cell line (BT2C) showed low phagocytic activity, and almost no destruction was observed in the border zone between tumor cells and normal tissue. Phagocytosis seems to be an inherent property of both normal and malignant glial cells, although the fraction of phagocytic cells varies from one cell line to another. In organ culture high phagocytic capacity of invasive glioma cells seems to be related to destructive activity on the normal brain tissue during invasion.

Animals↗
PubMed1987

DNA content and chromosomes in permanent cultured cell lines derived from malignant human gliomas.

DNA content, as determined by flow cytometry (FCM), and ploidy, as determined planimetrically from chromosomes, were measured for 17 established human glioma-derived cell lines. Values obtained by these methods corresponded well for two of two near-diploid lines, two of two hyperdiploid-hypotriploid lines, four of four hypertriploid-hypotetraploid lines, two of six hypertetraploid-hypopentaploid lines, the one hyperpentaploid line and two of two multiclonal lines. For the remaining four lines, ploidy values obtained by karyotyping were more than 10% lower than those obtained by FCM. Since karyotypic ploidy was determined by planimetric measurements of the chromosomes, which corrects for deviations in chromosome size, the lower values could not be explained by large marker chromosomes. Technical problems, such as random chromosomal loss in karyotypic preparations, adherent bits of cytoplasm in nuclear preparations for FCM or differences in nuclear stainability, are possible reasons for the discrepancies. Alternatively, chromosomes in some glioma cell lines may actually contain an increased amount of DNA. FCM and karyotyping provide complementary information in the initial evaluation of cultured cell lines. For sequential studies, such as are used for monitoring cultured lines, the rapidity of FCM makes it the more practical method.

Cell Line↗
PubMed1987

Transmission and scanning electron microscopy of N2 microbubble-activated human platelets in vitro.

When N2 microbubbles are stirred in platelet-rich plasma, they cause a fall in the number of free platelets. Changes in the platelets ultrastructure during this interaction between gas bubbles and platelets have been studied by transmission and scanning electron microscopy. Platelets, and aggregates of platelets, adhere to the surface of the N2 microbubbles. This adhesion induces ultrastructural changes (shape change, pseudopod formation, granule centralization, fusion, and disappearance) that are similar to "classical" agonists like ADP, collagen, and thrombin. These ultrastructural studies further strengthen our previous contention that N2 microbubbles activate platelets in a way similar to these physiologic agonists, and show that the previously reported fall in the number of free platelets is due to platelet aggregation. Platelet aggregates are also present in the interstices between gas bubbles. Fixed N2 microbubbles have been demonstrated, and these can be broken like cracked eggs by means of the electron beam in the electron microscope. Possible mechanisms for activation of the platelets by the gas bubbles are: through ADP released from some few platelets; by diffusion of gas after bubble-platelet interaction; and through certain fractions of the plasma proteins and lipids in the bubble surface that may act as binding sites for the platelets and promote adhesion and spreading of the platelets over the surface.

Blood Platelets↗