Janet L. Gross

MORPHOLOGIC, IMMUNOLOGIC, BIOCHEMICAL, AND CYTOGENETIC CHARACTERISTICS OF THE HUMAN GLIOBLASTOMA-DERIVED CELL LINE, SNB-19

SummaryHuman glioma-derived cell cultures and lines have proven to be of significant value in the study of the basic properties that contribute to the highly malignant, invasive and angiogenic phenotype of glioblastoma multiforme tumors. It is frequently difficult to establish lines that retain glial tumor properties in long term culture. The SNB-19 cell line has maintained and exhibited properties of transformation, differentiation, autocrine growth response, and tumorigenesis while remaining in culture for over 13 yr and undergoing over 200 passages. This human line has been utilized in a wide range of studies related to the basic properties of human glioblastoma multiforme. In this report, we summarize the immunologic, biochemical, and cytogenetic properties of this versatile cell line and its utility for additional mechanistic investigation into the pathophysiology of the progression of human malignant gliomas.

Characterization of specific [3H]dimethylstaurosporine binding to protein kinase C

The microbial alkaloid staurosporine is a member of a recently described family of protein kinase inhibitors. [N,N-dimethyl-3H]N-dimethylstaurosporine ([3H]DMS) was prepared from staurosporine by methylation with [3H]methyl iodide. Since staurosporine inhibits protein kinase C (PKC) most potently, the binding of [3H]DMS to this enzyme was examined. Unlike [20-3H(N)]phorbol-12,13-dibutyrate ([3H]PDBu) binding to PKC, [3H]DMS binding was not calcium or phosphatidylserine (PS) dependent. Binding was reversible, with a T1/2 of 69 min and a Koff of 0.01/min. Non-specific binding was defined by a 500-fold molar excess of staurosporine and was less than 10% of total [3H]DMS binding. Specific binding of [3H]DMS was consistent with a single class of binding sites with a Kd of 3.8 +/- 0.6 nM and a Bmax of 675 +/- 30 pmol/g tissue. In competition experiments, staurosporine inhibited [3H]DMS binding with a Ki of 4.7 +/- 0.6 nM, indicating that the two alkaloids had a similar potency for PKC. Also, unlabeled DMS and staurosporine inhibited [3H]DMS binding and PKC catalysis with equivalent potencies. Highly purified rat brain PKC bound equimolar amounts of [3H]PDBu and [3H]DMS. In contrast, crude rat brain PKC, which had been proteolysed to generate a PS and Ca2+ independent enzyme (PK-M) retained the ability to bind [3H]DMS, but not [3H]PDBu. In addition, the kinase inhibitors K-252a and H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] inhibited [3H]DMS binding, whereas PDBu did not. These results indicate that [3H]DMS is a useful ligand to identify catalytic inhibitors of kinase activity and to explore their mechanisms of action.

Evaluation of a novel bis-naphthalimide anticancer agent, DMP 840, against human xenografts derived from adult, juvenile, and pediatric cancers

The new bis-naphthalimide antitumor agent (R,R)2,2′-[1,2-ethanediylbis[imino(1-methyl-2.1-ethanediyl)]-bis {5-nitro-1H-benz[de]-isoquinoline-1,3-2H) dione} dimethanesulfonate (DMP 840) was evaluated against parental and multidrug-resistant human KB cell lines in vitro and against these lines growing as xenografts in immunedeprived mice. In vitro, KB8-5 cells were 50-fold resistant to vincristine but only 16-fold resistant to DMP 840 as measured by clonogenic survival. For in vivo evaluation, DMP 840 was given by i. v. injection daily for 9 days or for 5 days/week for 2 consecutive weeks [(dx5)2]. In contrast to the cross-resistance of KB cell lines in vitro, both KB3-1 and KB8-5 tumors were highly and equally sensitive to DMP 840; only KB3-1 xenografts demonstrated sensitivity to vincristine, which was consistent with the in vitro results. DMP 840 was also evaluated against a panel of human tumors comprising colon adenocarcinoma and rhabdomyosarcoma xenografts. Against eight lines of colon adenocarcinoma, DMP 840 caused a high frequency of partial and complete regressions in two lines and significant inhibition of growth in two lines. DMP 840 caused complete regressions in five of six lines of advanced rhabomyosarcomas, demonstrating a broad range of effective dose levels. The pattern of activity against this tumor panel was similar but not identical to that of two inhibitors of topoisomerase I. There was no cross-resistance to DMP 840 in xenografts selected for resistance to vincristine or in a rhabdomyosarcoma selected for resistance to the topoisomerase I inhibitor topotecan. In contrast, a colon tumor selected for topotecan resistance was completely resistant to DMP 840. Slight cross-resistance to DMP 840 was demonstrated in a rhabdomyosarcoma xenograft that was selected for primary resistance to melphalan and was cross-resistant to topoisomerase I inhibitors. The pattern of activity and cross-resistance in these tumors was compared with that shown by two agents that inhibit topoisomerase I: topotecan and CPT-11.

Overview Oncologic, Endocrine & Metabolic: Recent advances in angiogenesis inhibitors

(1994). Overview Oncologic, Endocrine & Metabolic: Recent advances in angiogenesis inhibitors. Expert Opinion on Therapeutic Patents: Vol. 4, No. 6, pp. 641-654.

Protein kinase C-linked inactivation of the interleukin-1 receptor in a human transformed B-cell line.

The effect of tumor-promoting phorbol ester treatment on the binding of interleukin-1 beta (IL-1 beta) to specific cell surface receptors was investigated. A 1 h exposure of Raji human B lymphoma cells with the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu), reduced IL-1 beta binding by up to 90% of control cells. This effect was dose-dependent and was not observed with 4-alpha-phorbol, an inactive tumor promoter. Analysis of 125I-labeled IL-1 beta binding to intact cells revealed that PDBu caused a 91% decrease in high-affinity cell-surface receptor number without an effect on receptor affinity. The phorbol ester response was rapid (30 min), observed both at 4 and 37 degrees C, and was preceded by the rapid translocation (t much less than 6 min) of protein kinase C (PKC) from the cytosol to the cell membrane. The PDBu-induced decrease in IL-1 beta receptor number was inhibited by prior incubation of cells for 30 min with the PKC inhibitor 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine (H7). The decrease in receptor binding was not due to enhanced IL-1 beta receptor internalization or shedding into the extracellular medium, since a similar effect was observed with solubilized IL-1 beta receptor. The most likely explanation for the phorbol ester effect appears to be cell surface inactivation of IL-1 receptors. These data suggest that modulation of PKC activity could play a role in the regulation of the IL-1 beta receptor.

Tumor growth regulation by modulation of basic fibroblast growth factor

Fibroblast growth factors (FGF) were initially characterized for their mitogenic properties [1]. Since then, members of this family of growth factors have been shown to be growth stimulatory for a variety of cell types in vitro, including glial cells [2–4]. The importance of FGF in tumor biology is supported by the demonstration that FGFs stimulate tumor angiogenesis [5], a process critical for the sustained growth of solid tumors [6]. In addition, cells transfected with bFGF exhibit amplified autocrine growth in monolayer culture and in soft agar, suggesting a role in cell transformation [7]. Finally, several recently described oncogenes, int-2, hst/KS3, and FGF-5, discovered in human tumors encode proteins which share 40–45% homology with bFGF [8–11].

Inhibition of angiogenesis as a strategy for tumor growth control

Angiogenesis is a complex sequence of events leading to the formation of new capillaries. Although essential to maturation and wound healing, most angiogenesis in the adult is associated with pathological events, such as the development of solid tumors. One approach to the inhibition of angiogenesis is the antagonism of basic fibroblast growth factor, a major angiogenic protein. Evidence is reviewed to suggest that inhibiting angiogenesis results in the suppression of tumor growth.

Modulation of synovial fibroblast plasminogen activator and plasminogen activator inhibitor production by protein kinase C

Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA).

Angiosuppressive and Antiproliferative Actions of Suramin: A Growth Factor Antagonist

Suramin is a novel anticancer agent1 that appears to be effective against advanced adrenocortical carcinoma2,3, prostatic cancer4,5, ovarian cancer6, renal cell carcinome and certain refractory lymphomas8. The antiproliferative action is possibly related to the ability of suramin to block the binding of autocrine growth factors to their receptors9-18, to inhibit a variety of cytoplasmic and intranuclear enzymes critical for cell maintenance and proliferation19,20, and to disrupt cellular respiration and energy balance21. Cell migration and adhesion of B16 melanoma cells to the extracellular matrix is inhibited following suramin exposure 22,23 suggesting a mechanism for suramin to inhibit tumor invasion.

Binding sites for basic fibroblast growth factor on solid tumors are associated with the vasculature

Basic fibroblast growth factor (bFGF) is a member of a family of heparin binding growth factors that exhibit mitogenic and proliferative effects on many mesoderm-and ectoderm-derived cells [1, 2]. It has been shown to be a major angiogenic factor involved in the neovascularization of development and wound healing, as well as the abnormal proliferation of vasculature associated with tumor growth, metastases, diabetic retinopathy and atherosclerosis [3–5]. Multiple forms of the protein have been shown to exist and multiple classes of receptors have been identified [6].