Eirik Helseth

CONCOMITANT EXPRESSION OF HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR AND THE RECEPTOR C-MET IN HUMAN MYELOMA CELL LINES

Myeloma cell line supernatants were screened for their ability to inhibit the activity of transforming growth factor-β (TGFβ) in the mink lung cell (Mv-1-Lu) bioassay. Supernatant from the human myeloma cell line JJN-3 contained potent TGFβ antagonistic activity. This activity was isolated and found to be associated with a 72-78-kDa glycoprotein. Specific polyclonal and monoclonal antibodies were generated toward the 72-78-kDa protein, and these antibodies precipitated the TGFβ inhibitory activity from JJN-3 supernatant. Upon amino acid sequencing the protein appeared to be identical to hepatocyte growth factor (HGF), and some of the generated antibodies directly blocked the action of recombinant HGF in various assays. By HGF-specific polymerase chain reaction we demonstrated that HGF mRNA was expressed in five out of five tested myeloma cell lines. The level of HGF protein in supernatants showed great variation from >500 ng/ml in JJN-3 supernatant to a few ng/ml in the supernatants from other myeloma cell lines. The same five cell lines were also screened for expression the HGF receptor c-MET. Four of them expressed the receptor as shown by reverse transcriptase-polymerase chain reaction and Western blot. The receptor was shown to be constitutively phosphorylated in the human myeloma cell line JJN-3. This receptor could be dephosphorylated by anti-HGF antibodies, indicating the existence of an autocrine HGF loop in this cell line. We propose that HGF/c-MET may play a role in multiple myeloma.

Relationships between Ki-67 labelling index, amplification of the epidermal growth factor receptor gene, and prognosis in human glioblastomas.

SummaryThe aim of this study was to determine possible relationships between Ki-67 labelling index (Ki-67 LI), amplification of the epidermal growth factor receptor (EGFR) gene, and prognosis in human glioblastomas. Ki-67 LI was determined on cryosections of biopsy specimens of 20 human glioblastomas with a mouse antihuman Ki-67 monoclonal antibody. Amplification of the EGFR gene was determined by slot blot and Southern blot analyses of DNA extracted from the tumour biopsies. The Ki-67 LI was higher in the glioblastoma group with EGFR gene amplification (8 tumours, median value of Ki-67 LI 4.2, range 0.4–24.6) than in those without EGFR gene amplification (12 tumours, median value of Ki-67 LI 0.8, range 0.2–11.8) (0.05 p<0.1). The glioblastoma patients with Ki-67 LI>1.5 (10 tumours) had a statistically significant shorter survival than those with Ki-67 LI<1.5 (10 tumours) (p<0.05). The glioblastoma patients with EGFR gene amplification lived shorter time than those without EGFR gene amplification (p>0.05).

Amplification of the Epidermal Growth Factor Receptor Gene in Biopsy Specimens from Human Intracranial Tumours

Amplification and overexpression of proto-oncogenes are associated with the malignant nature of some human tumours. In this study we have determined the prevalence of amplification of the proto-oncogenes c-erb B1 (= epidermal growth factor receptor gene), c-erb B2 and c-myc in 44 human intracranial tumours (27 gliomas, six metastases to the brain and 11 meningiomas). None of the tumours had an amplified c-erb B2 gene and only two tumours had an amplified c-myc gene. Nineteen per cent (five out of 27) of the gliomas, 50% (three out of six) of the brain metastases and 0% (0 out of 11) meningiomas had an amplified EGF-receptor gene. Amplification of the EGF-receptor gene appeared to give a growth advantage when single-cell suspensions of the tumours were grown in agarose.

Effects of type beta transforming growth factor in combination with retinoic acid or tumor necrosis factor on proliferation of a human glioblastoma cell line and clonogenic cells from freshly resected human brain tumors.

SummaryType beta transforming growth factor (β-TGF) is a potent regulator of cell growth and differentiation. The human glioblastoma cell line, T-MGl, was growth inhibited by β-TGF under anchorage independent conditions. The antiproliferative effect of β-TGF was potentiated to nearly total arrest by low doses of retinoic acid (RA) or tumor necrosis factor (TNF), while epidermal growth factor, platelet-derived growth factor, interleukin-2, and gamma interferon did not have this potentiating effect. The potentiation of the β-TGF effect by RA and TNF could not be explained by modulation of the epidermal growth factor receptor, the β-TGF receptor, or the TNF receptor. β-TGF alone and in combination with RA or TNF were further tested on primary cultures from freshly resected human glioma biopsies (n=13). There was great individual variation in sensitivity to β-TGF, RA, or TNF. The astrocytoma and oligodendroglioma cells were inhibited to various degrees by β-TGF or TNF, while most of the glioblastomas were not sensitive to these agents. Most of the biopsies were stimulated by RA. RA or TNF did not potentiate the growth inhibitory effect of β-TGF on biopsy cells. We therefore think it unlikely that β-TGF in combination with RA or TNF will be effective agents in the treatment of gliomas.

The effects of type beta transforming growth factor on proliferation and epidermal growth factor receptor expression in a human glioblastoma cell line

SummaryType beta transforming growth factor (B-TGF) is a potent growth inhibitor to many human tumor cell lines. Very little is known about the mechanism for this growth inhibitory action of BTGF We here report the effect of B-TGF on proliferation and epidermal growth factor receptor (R-EGF) expression in a human glioblastoma cell line named T-MG1.B-TGF inhibite the soft agar growth of T-MG1 cells. Maximum inhibition was 70%, achieved with 0.5 units BTGF. BTGF had no effect on monolayer growth of T MGl cells.T-MG1 cells contained abundant R-EGF, which could be divided into two subpopulations, one high affinity and one low affinity population of R-EGF. Treatment with B-TGF caused an initial decrease (0-6 h) in EGF-binding, followed by an increase in EGF-binding which reached maximum after 24 h exposure to B-TGE. Since addition of EGF to agar cultures gave no additional increase in inhibition by B-TGF and EGF alone had no inhibitory effect, we believe that binding of EGF to its receptor is not part of the pathway mediating the inhibitory effect of B-TGF.All neoplastic cells have lost some measure of growth control and the cellular elements involved are growth factors, growth factor receptors and oncogenes. T-MG1 cells contain abundant R-EGF and this may partly explain their malignant nature (malignant nature is here defined as ability to proliferate in agarose). Type alpha transforming growth factors, which in some cancer cells act as uncontrolled autocrine growth factors, were not found in protein extracts from T-MG1 cells. BTGF, which is normally found in all cells, could not be detected in protein extracts from T-MG1 cells. The lack of B-TGF as an autocrine growth inhibitor may be of importance for maintaining the malignant nature of T-MG1 cells.

Tumor necrosis factor-α regulates mRNA for urokinase-type plasminogen activator and type-1 plasminogen activator inhibitor in human neoplastic cell lines

Tumor necrosis factor-alpha (TNF-alpha) was found to induce type-1 plasminogen activator inhibitor (PAI-1) antigen in the human fibrosarcoma cell line HT-1080, and PAI-1 and urokinase-type plasminogen activator (u-PA) antigens in the human carcinoma cell line T-CAR1; tissue-type plasminogen activator (t-PA) antigen was not affected or slightly decreased. The effects in HT-1080 and T-CAR1 cells were preceded by increases in the cellular levels of the corresponding mRNAs. Cycloheximide caused an increase of PAI-1 mRNA in T-CAR1 cells, but not in HT-1080 cells; during this increase the relative abundance of the two PAI-1 mRNA species, of 2.3 kb and 3.4 kb, respectively, changed strongly in favor of the longer transcript. We conclude that TNF-alpha may affect proteolytic activity in the microenvironment of cells in malignant tumors by affecting gene expression of u-PA and PAI-1.

Effects of interferon-gamma and tumor necrosis factor-alpha on clonogenic growth of cell lines and primary cultures from human gliomas and brain metastases

In this study we tested the effect of interferon‐gamma (IFN‐gamma), tumor necrosis factor‐alpha (TNF‐alpha) and a combination of these immunotherapeutics on clonogenic growth of cell lines and primary cultures of malignant human brain tumors. Out of 29 primary cultures studied, 5 were inhibited 50% or more by IFN‐gamma (10 ng/ml), 6 were inhibited 50% or more by TNF‐alpha (10 ng/ml) and 14 were inhibited 50% or more by a combination of IFN‐gamma (10 ng/ml) and TNF‐alpha (10 ng/ml). The doses of the immunotherapeutics used in this in vitro study are achievable in serum after intravenous administration of IFN‐gamma and TNF‐alpha without causing unacceptable side effects. We believe that some glioma patients and some patients with brain metastases will benefit from receiving treatment with IFN‐gamma, TNF‐alpha or a combination of these immunotherapeutics. The patients should be selected for treatment with these immunotherapeutics according to in vitro sensitivity results.

Overexpression of the epidermal growth factor receptor gene in a human carcinoma cell line, derived from a brain metastasis.

SummaryAbnormally high expression of epidermal growth factor receptors (EGF-receptors) may contribute to the unregulated growth of some tumors. We here report the EGF-receptor numbers and the effects of epidermal growth factor (EGF) on two human cell lines. The glioblastoma cell line T MGl had 135 000 EGF-receptors per cell, was slightly growth stimulated by EGF and showed no obvious change in morphology after exposure to EGF. The carcinoma cell line TCARI, derived from a brain metastasis of a carcinoma of the adrenal cortex, had approximately 7 million EGF-receptors per cell. EGF had a significant antiproliferative effect on these cells and caused rounding and detachment of cells in adherent cultures. The cell lines may become useful in future studies concerning the role of the EGF-receptors in malignant growth.

Polysomy of chromosome 7 is associated with amplification and overexpression of the EGF‐receptor gene in a human carcinoma cell line derived from a brain metastasis

Overexpression of the EGF‐receptor gene is associated with the malignant nature of some tumors. We have recently reported the establishment of a human carcinoma cell line (T‐CAR1), derived from a brain metastasis, that had 7 million EGF receptors per cell and was growth inhibited by EGF. The present study was carried out in order to furhter characterize the EGF‐receptor protein in T‐CAR1 cells, and to see if the overexpression of the EGF‐receptor gene in these cells was associated with abnormalities at the genomic level. We have compared the T‐CAR1 cells with the human glioblastoma cell line T‐MG1, which has 135,000 EGF‐receptors and is growth stimulated by EGF. The MW of the EGF receptors in T‐CAR1 cells and T‐MG1 cells was estimated to be 170 kDa, equal to the normal EGF‐receptor. However, in T‐CAR1 cells an additional protein reacted with the monoclonal antibody directed against the internal domain of the EGF receptor. The levels of EGF receptor‐related RNAs in T‐CAR1 cells and T‐MG1 cells reflected the number of EGF receptors in these cell lines. The EGF‐receptor gene was amplified ten‐fold in T‐CAR1 cells, while it was not amplified in T‐MG1 cells. No restriction fragment length polymorphism of DNA digested with various restriction enzymes was seen in either of the cell lines. Chromosomal analysis of T‐CAR1 cells showed polysomy of chromosome 7 and marker chromosomes derived partly from chromosome 7. Thus, in the T‐CAR1 cell line it was an association between polysomy of chromosome 7 and EGF‐receptor gene amplification.

An isotope agarose assay for rapid testing of the sensitivity of glioma biopsies to chemotherapeutic drugs and biological response modifiers. Effects of BCNU, vincristine, lymphokines and the recombinant agents interferon alpha 2c, interferon gamma and tumour necrosis factor.

SummaryA method based on growth of cells from glioma biopsies in a triple layer agarose system has been used to test the sensitivity of the tumour cells to different chemotherapeutic and immunotherapeutic agents. A special agarose that could be melted at 65°C, enabled registration of proliferation by an easy and precise thymidine incorporation technique.The well known concept that agarose permits proliferation of malignant cells and inhibits growth of benign cells was confirmed in this assay by using the malignant glioma cell lines T-MG 1 and U 251 MG, and the benign glia cells T-BG2 and T-BG3.All of the 15 glioma biopsies grew exponentially during the first 7 days. The correlation between thymidine incorporation on day 7 and colony number on day 14 was very good for the glioma cell line (r=0.96) and also for the glioma biopsies (r=0.89), indicating that the sensitivity evaluation can be done as early as 7 days after the operation.Lymphokines, made by BCG-stimulated lymphocytes, had a strong inhibitory effect on the growth of some glioma biopsies, while others were only slightly inhibited or even stimulated. There was also a huge variation in the sensitivity of the biopsies to BCNU, but the sensitivity pattern was completely different for BCNU and lymphokines. rIF-A had a moderate inhibitory effect on growth of the biopsies, while rTNF had a weak inhibitory effect. The response to rIF-G varied from stimulation of some biopsies to strong inhibition of others. There was no similarity in the sensitivity pattern of the biopsies to rIF-A and rIF-G. The combination of rIF-G and rTNF had a synergistic inhibitory effect.