Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

Establishment of a human glioma cell line bearing a homogeneously staining chromosomal region and releasing α- and β-type transforming growth factors

Summary A human glioma cell line (YKG1), which was positively identified for glial fibrillary acidic (GFA) and S-100 proteins, was established from a surgical specimen of a patient with glioblastoma. Chromosome analysis of the cells revealed a homogeneously staining region (HSR) on a marker chromosome. The assay for transforming growth factors (TGFs) in the conditioned medium of the cell line revealed that it contained high levels of - and -type TGFs, which might regulate the growth of glioblastoma and influence on the peritumoral tissues.     

Protective actions of human recombinant basic fibroblast growth factor on MPTP-lesioned nigrostriatal dopamine neurons after intraventricular infusion

Basic fibroblast growth factor (bFGF, FGF-2) is a trophic factor for neurons and astrocytes and has recently been demonstrated in the vast majority of dopamine (DA) neurons of the ventral midbrain of the rat. Potential neuroprotective actions of FGF-2 in the l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) model have also been reported. The actions of the FGF-2 have now been further analyzed in a combined morphological and behavioural analysis in the MPTP model of the adult black mouse, using a continuous human recombinant FGF-2 (hrFGF-2) intraventricular (i.v.t.) administration in a heparin-containing (10 IU heparin/ml) mock cerebrospinal fluid (CSF) solution. Tyrosine hydroxylase (TH) immunocytochemistry in combination with computer assisted microdensitometry demonstrated a counteraction of the MPTP-induced disappearance of neostriatal TH-immunoreactive (ir) nerve terminals following the FGF-2 treatment. Unbiased estimates of the total number of nigral TH ir neurons, using stereological methods involving the optical disector (Olympus), showed that the MPTP-induced reduction in the number of nigral TH ir nerve cell bodies counterstained with cresyl violet (CV; by 56%) was partially counteracted by the FGF-2 treatment (by 26%). The behavioral analysis demonstrated an almost full recovery of the MPTP-induced reduction of the locomotor activity after FGF-2 treatment. This action was maintained also 1 week after cessation of treatment. The hrFGF-2 produced an astroglial reaction as determined in the lateral neostriatum and in the substantia nigra (SN) far from the site of the infusion, indicating that the growth factor may have reached these regions by diffusion to activate the astroglia. Immunocytochemistry revealed FGF-2 immunoreactivity (IR) in the nuclei of the astroglia cell population in the dorsomedial striatum and the microdensitometric and morphometric evaluation demonstrated an increase in the number, but not in the intensity, of these profiles on the cannulated side, suggesting the possibility that hrFGF-2 stimulates FGF-2 synthesis in astroglial cells with low endogenous FGF-2 IR. These results indicate that hrFGF-2, directly and/or indirectly via astroglia, upon i.v.t. infusion exerts trophic effects on the nigrostriatal DA system and may increase survival of nigrostriatal DA nerve cells exposed to the MPTP neurotoxin.     

Role of the stromal microenvironment in regulation of bone-marrow hematopoiesis after administration of dipyridamole

I. P. Pavlov First Leningrad Medical Institute. Central Roentgeno-Radiological Research Institute, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 7, pp. 98–100, July, 1990.     

Malignant progression of B16 melanoma cells induced in vitro by growth factors produced by highly malignant cells

Four mouse B16 melanoma subclones (G3.15, G3.5, G3.12 and G3.26) exhibit progressively greater growth capacity in vitro and in vivo. Previously, non-metastatic G3.15 cells were sequentially converted, in monolayer cultures, to the moderately-metastatic G3.5 cells, and then to a highly-metastatic G3.5^* phenotype. Both conversions were induced by hypoxia followed by confluence, and also occurred in tumors. G3.5^* cells were comparable with, yet distinguishable from, G3.12 cells in being growth-autonomous in culture. In this study, the presumption that rapidly-growing G3.26 cells represented the ultimate progression step in this clonal system was examined. Both G3.12 and G3.5^* cells converted in vitro to the G3.26 phenotype during growth in serum-free medium conditioned by G3.26 cell growth. By selective filtration of conditioned medium and characterization of the stability of growth- and conversion-promoting activities, three distinct activities were found to promote a two-step G3.12 to G3.26 phenotype conversion: (1) a < 10 kDa filtrate stimulated slight attachment and proliferation of G3.12 cells, effects that were reversible, partly attributable to accumulated lactate, and fully mimicked by medium acidification to pH 6.5; (2) medium acidification, together with a heat- and acid-stable but partially trypsin-sensitive > 10 kDa activity, induced G3.12 G3.5^* conversion that resulted in acquisition of growth autonomy; and (3) a heat-, acid- and trypsin-sensitive > l0 kDa activity induced G3.5^* G3.26 conversion, characterized by anchorage-independent growth in soft agar, and potent lung colonization following intravenous injection. Phenotype analysis of G3.12 tumors and lung metastases revealed that G3.5^*-like cells were regularly present in tumors and metastases, whereas G3.26-like cells occurred almost exclusively in large lung metastases. While G3.12 cells might convert to G3.5^* cells in order to disseminate, G3.26 cells are apparently not involved in metastatic spread but probably account for the rapid growth of established metastases.     

Keratinocyte-derived growth factors play a role in the formation of hypertrophic scars

In predisposed individuals, wound healing can lead to hypertrophic scar or keloid formation, characterized by an overabundant extracellular matrix. It has recently been shown that hypertrophic scars are accompanied by abnormal keratinocyte differentiation and proliferation, and significantly increased acanthosis, compared with normal scars. This study addressed the question of whether the development of normal and hypertrophic scars is regulated by differences in the growth factor profiles of both the epidermis and the dermis. The presence of interleukin-1alpha (IL-1alpha), IL-1beta, tumour necrosis factor-alpha (TNF-alpha), platelet-derived growth factor (PDGF), transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) was investigated in biopsies taken from breast reduction scars at 3 and 12 months following surgery. The samples were analysed by immunohistological methods and categorized as scars that remained hypertrophic (HH), became normal (HN) or remained normal after 12 months (NN). The epidermal expression of IL-1alpha was significantly increased in NN scars compared with HN and HH scars 3 and 12 months following operation, whereas the dermal expression showed no difference. PDGF was significantly increased in the dermis of normal scars after 3 months and in both the epidermis and the dermis of hypertrophic scars after 12 months. IL-1beta, TNF-alpha, TGF-beta and bFGF showed no differences. It is hypothesized that impaired production of keratinocyte-derived growth factors, such as IL-1alpha, leads to a decrease in the catabolism of the dermal matrix, whereas augmented epidermal PDGF production leads to increased formation of the dermal matrix in hypertrophic scars. These observations support the possibility that the epidermis is involved in preventing the formation of hypertrophic scars.     

Social isolation stress augments angiogenesis induced by colon 26-L5 carcinoma cells in mice

We have previously shown that tumor necrosis factor-α (TNF-α), which is an important angiogenesis-related factor, was over-secreted in male BALB/c mice under social isolation stress as compared with the control, and closely associated with a remarkable elevation of tumor invasion and metastasis of colon 26-L5 carcinoma cells. In the present study, we explored the effect of isolation stress on the angiogenesis caused by colon 26-L5 carcinoma cells in vivo and in vitro. Social isolation lead to the enhancement of tumor growth after intrahepatic implantation with a fragment of colon 26-L5 tumor. Angiogenic response (number of vessels oriented towards tumor mass) and tumor growth (size) were significantly increased in the socially isolated mouse relative to that in the group-housed mice. Furthermore, higher protein level of hepatic TNF-α was found in the stressed mice than that in the control. Expression of mRNA for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were also elevated in the tumor regions and liver tissues of the stressed mice in comparison with that in group-housed mice. On the other hand, hepatic sinusoidal endothelial (HSE) cells treated with TNF-α exhibited a marked promotion of the migration, invasion, expression of mRNA for matrix metalloproteinase (MMP)-9, and tube-like formation, but no cytotoxicity against the cells in vitro. The above data suggest that the social isolation stress augmented the tumor-induced angiogenesis probably by up-regulating the angiogenesis-related factors, including TNF-α, VEGF and HGF, and consequently mediating the functions of endothelial cells such as migration, invasion, and tube-like formation.     

Transforming growth factor J3 type II receptor (TGFpRII) mutation in gastric lymphoma without mutator phenotype

A new mutation in the serine-threonine klnase domain of the transforming growth factor β type II receptor (TGFpRII) was found in a case of diffuse, B cell non-Hodgkin's lymphoma of the stomach. A mfssense mutation (ACA to GCA, Thr to Ala) was detected In exon 5, and a wild type allele was also present. This Is the first naturally occurring mutation in the klnase domain of this gene identified in human primary lymphoma. The replication error at three loci was negative, and the poly A tract of exon 3, which is frequently a target of mismatch repair genes, was intact. Malignant lymphoma of B cell origin in the stomach Is an addition to an expanding catalogue of tumors with TGFβRII alterations, and the biological sequelae of the change in the functional domain and the clinical characteristics of the patient in this study are intriguing.     

Enhanced production of transforming growth factor-beta (TGF-beta) during autologous mixed lymphocyte reaction of systemic sclerosis patients.

Systemic sclerosis (SSc) is characterized by systemic fibrosis and microvascular lesions. As TGF-beta is suggested to be related to skin fibrosis, we examined the production of TGF-beta from peripheral mononuclear cells (MNC) of SSc patients. Since anti-TGF-beta neutralizing antibody improved the defective proliferative response in autologous mixed lymphocyte reaction (AMLR) of SSc patients, TGF-beta was thought to participate in the decreased AMLR of SSc patients. Greater amounts of TGF-beta in the active as well as in the latent forms were produced during AMLR of SSc patients than that of normal subjects. It was suggested that TGF-beta excessively produced from the MNC of SSc patients might play a major role in the fibrosis of the patients during AMLR-like in vivo responses.     

内痔粘膜及血管上皮细胞VEGF/FGF2的表达与内痔分期的相关性分析

目的 通过观察人体内痔不同分期粘膜及血管内皮细胞生长因子（VEGF）及碱性成纤维细胞生长因子（FGF2）的表达，探讨内痔的发生及发展机制。 方法 收集南方医院肛肠科门诊手术切除的Ⅰ、Ⅱ、Ⅲ期内痔标本134例（Ⅰ期42例，Ⅱ期45例，Ⅲ期47例），内痔周围正常肠壁组织40例作为对照，采用HE染色观察组织的病理学变化，采用免疫组织化学方法检测血管内皮细胞VEGF及FGF2的表达。 结果 正常组及Ⅰ期内痔黏膜层被覆上皮完整，未见扩张血管；Ⅱ期内痔黏膜层被覆上皮破坏，黏膜肌层破坏，黏膜层内见新生血管；Ⅲ期内痔黏膜层被覆上皮破坏，见血管管壁增厚迂曲，管腔扩张；与正常粘膜成纤维细胞相比VEGF在粘膜层成纤维细胞表达水平明显升高，并随分期增高而增高（F=883.961，P<0.01），FGF2也存在相同表达（F=656.013，P<0.01）；与正常组相比VEGF在血管内皮细胞表达水平明显升高，并随分期增高而增高（F=776.561，P<0.01），FGF2在血管内皮细胞的表达水平存在相同趋势（F=1066.458，P＜0.01）。 结论 VEGF及FGF2在内痔的形成过程中具有促进血管内皮细胞和粘膜下成纤维细胞增生的作用，同时可作为内痔发生发展的分子标志物。