Differential in vitro effects of chemotherapeutic agents on primary cultures of human ovarian carcinoma.

The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process.

Measurement of NK activity by the microcytotoxicity assay (MCA): a new application for an old assay.

Natural killer (NK) cells are spontaneously cytotoxic immune effector cells with the ability to selectively destroy tumor cells without harming normal cells. To perform this function, NK cells utilize two main cytotoxicity pathways, the well known perforin/granzyme-mediated secretory/necrotic killing and the recently defined TNF family ligand-mediated non-secretory/apoptotic killing. The former mechanism is manifested mainly against a few cultured leukemia cell targets, while the latter mediates killing against a large variety of tumor cell targets. Therefore, the biological role and significance of these mechanisms might be different. The NK cell-mediated necrotic killing has been reliably and selectively measured in humans by the standard 4-h 51Cr release assay (CRA) against K562 myeloid leukemia cell targets. However, no standardized high throughput assay is available for testing the NK cell-mediated apoptotic killing. Here, we introduce the modified MCA as a convenient method for measuring perforin/granzyme-independent NK cell-mediated apoptotic killing. The assay is performed in microwells of Terasaki tissue culture microtest plates, using adherent tumor cell targets, which are selectively susceptible to non-secretory/apoptotic killing and resistant to secretory/necrotic killing mediated by NK cells. Target cells are plated in microwells and incubated overnight to adhere to the plastic surface and to regenerate cell surface-bound TNF family receptors. Following this adherence, target cells are co-incubated with freshly isolated human peripheral blood mononuclear leukocytes (PBMNL) or purified subpopulations of immune cells for 24 h in various effector/target (E/T) ratios. During this incubation, dead target cells become non-adherent and are removed by washing the wells. Remaining adherent (viable) target cells are fixed, stained and optically counted. A notable dose-dependent (peak at 200:1 E/T ratio), time-dependent (peak at 24 h of incubation) and donor-dependent killing of tumor cells was consistently and reproducibly induced by PBMNL of normal donors. Using purified subpopulations of immune cells, it was demonstrated that among PBMNL, CD3(-)CD56(+)CD16(+) mature NK cells are the only mediators of tumor cell killing in MCA, as well as in CRA. Comparative studies of NK activity detected by MCA and CRA, performed with PBMNL from normal individuals and breast cancer patients, showed no significant correlation between the cytotoxicities measured in the two assays. In addition, while NK activity measured in CRA was normal in most breast cancer patients, NK activity assessed in MCA was decreased in a large majority of the patients. Thus, MCA is a sensitive NK assay, which is biologically different from CRA, and may be clinically relevant. MCA has also a higher throughput, and is more practical and economical than CRA.

Hepatocyte growth factor (HGF) and receptor (c-met) in normal and malignant astrocytic cells.

BACKGROUND: Hepatocyte growth factor (HGF) is a multifunctional peptide that binds to a specific receptor, c-met. Both HGF and c-met have been identified in normal brain and on glial tumors. The purpose of this study is to further define the biologic importance of HGF and c-met on normal and malignant glial cells grown in vitro. MATERIALS AND METHODS: Nine human malignant glioma-derived tumor cell cultures and cultures of astrocytes derived from normal brain were examined for c-met and HGF transcripts using Northern blot or RT-PCR analysis. Cellular invasiveness was quantitated by mechanical assay and mitogenesis was determined by cell count. RESULTS: C-met was expressed in five of seven malignant glioma-derived tumor cell cultures and in both normal astrocyte cultures. HGF transcript was not detected in any of the cell cultures. HGF supplementation enhanced invasiveness in c-met positive cell lines and did not alter cellular mitogenesis in the assayed cultures. CONCLUSIONS: These findings suggest that HGF is a potent stimulator of invasiveness in c-met positive malignant glioma-derived tumor cells and is not an active cytokine with regards to in vitro glial cell proliferation. HGF may therefore stimulate glioma cellular invasion in vivo through binding to its receptor and by activating tyrosine kinase secondary messengers.

Variability of intracellular concentrations of basic fibroblast growth factor in cultured human gliomas.

BACKGROUND: Basic fibroblast growth factor (bFGF) is a small peptide with angiogenic and mitogenic properties that supports the growth and proliteration of human malignant glial tumors in vitro and in vivo in an autocrine fashion. The purpose of this study was to examine the potential relationship between intracellular bFGF concentrations and grade of glial malignancy using a monolayer cell culture system. MATERIALS AND METHODS: Samples from 13 histopathologically verified astrocytic brain tumors and two non-tumorous astrocyte specimens were grown in tissue culture and examined both early and late after explantation together with a bFGF-producing reference cell line. RESULTS: Elevated intracellular concentrations of bFGF were noted in the reference line as well as two of five other glioblastoma multiforme-derived cell cultules, three of five anaplastic glioma-derived cell cultures, and two of three astrocytoma-derived cell cultures. The cells derived from non-tumorous astrocyte specimens expressed low concentrations of bFGF. CONCLUSION: This study demonstrates that overlap exists between the grade ot glial malignancy and intracellular bFGF levels.

Morphologic, immunologic, biochemical, and cytogenetic characteristics of the human glioblastoma-derived cell line, SNB-19.

Human 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.

The future of therapy for glioblastoma.

Effective therapy for malignant gliomas has centered on traditional approaches such as surgery and radiation therapy. Over the past two decades, more innovative approaches involving the use of chemotherapy and immunotherapy have been developed. Although these techniques have improved the quality of survival for many patients, the median survival following diagnosis and adjuvant treatment still remains only about a year. Recently, genetically engineered viruses for gene transduction and targeted cell killing have been used successfully in the experimental treatment of glioblastoma multiforme. We provide a review of the current and possible future therapies for malignant glioma with the belief that molecular biologic and genetic techniques offer the greatest hope of significantly altering the course of disease.

The effect of lipoproteins on human glioblastoma growth in vitro.

Experiments were performed using an established human glioblastoma cell line to determine the effect of lipoproteins on regulating their growth. It was found that synthetic and natural human high density lipoproteins (HDL) were effective in inhibiting tumor cell growth in a nontoxic, dose-dependent manner, and that the LD50 was 10-fold lower than that for normal rat astrocytes grown under identical conditions. In the presence of the antioxidant, glutathione, essentially all of the growth-inhibiting properties of HDL could be reversed suggesting that oxidized lipids from the HDL interacting with the plasma membranes of the glioblastoma cells were responsible for the growth-inhibiting effect observed. The markedly lower concentration of HDL required to inhibit glioblastoma cells in culture compared to normal astrocytes suggested that the mechanism of HDL-induced inhibition may be important for tumor growth in vivo. One possible mechanism under investigation is the possibility of HDL modulation of a membrane-associated, tumor-specific phosphatase.

Fragile beginnings: identification and treatment of postpartum disorders.

The transition to parenthood has long been recognized as a normal developmental crisis. However, 7 percent to 10 percent of all new mothers experience serious postpartum disorders that lie beyond the realm of the common "maternity blues." Postpartum depression and psychosis are hormonally based psychiatric illnesses that pose risks to the new mother and the family system. Too often, they are misdiagnosed as exclusively psychological and are undertreated by health care professionals. Early detection, assessment, and treatment are critical for the health and safety of both mother and child. Aggressive treatment may include individual and family therapy as well as medication. Women at particular risk for these illnesses should be identified during pregnancy. Efforts also should be made to prevent recurrence after subsequent deliveries.

Anatomy of the splanchnic circulation.

The blood supply to the intestines is a complex one, including branches of the three main splanchnic arteries as well as a vast collateral circulation. The variant anatomy and collateral pathways are described, based on anatomic dissections and angiographic studies, to focus attention on anatomically based explanations for clinical entities.

Basic fibroblast growth factor: a potential autocrine regulator of human glioma cell growth.

Basic fibroblast growth factor (bFGF) is a potent mitogen and angiogenic factor. bFGF is expressed by a variety of solid human tumors and has been implicated as an autocrine regulator of tumor growth. Different solid tumor lines including glioma, colon carcinoma and melanoma were examined for intracellular immunoreactive bFGF, high- and low-affinity bFGF receptors and mitogenic response to bFGF when grown in chemically defined medium. All tumor lines contained significant levels of bFGF. In addition, all tumor lines contained subsets of five forms of immunoreactive bFGF, as well as 0.68-20 x 10(6) low affinity bFGF binding sites (Kd = 15-300 nM). Most, but not all lines exhibited high affinity bFGF receptors (Kd = 25-40 pM). Glioma cell lines were distinguished by expressing the highest levels of bFGF protein as well as the most high-affinity receptors for bFGF. Furthermore, glioma cell lines were the only tumor type mitogenically responsive to bFGF. These results indicate that glioma cells express high levels of this potent mitogen and angiogenic factor relative to human colon carcinoma and melanoma cells. The expression of bFGF and bFGF receptors by glioma cells may be related to abnormal growth and neoplastic progression in these tumors.

Selective cytoplasmic and membrane changes induced by cisplatinum.

Cisplatinum (cis-dichlorodiammineplatinum II (NSC-119875], proven to be of therapeutic value in a variety of solid tumors, is thought to have DNA as its major target. Prior in vitro studies have suggested that it also induced cell membrane and cytoplasmic changes. To better understand glial tumor cell sensitivity to cisplatinum and to design more effective adjuvant therapy, three cisplatinum sensitive human glioma-derived cell lines, SNB-1, SNB-3, SNB-4, were examined by transmission electron microscopy for cisplatinum induced changes. Tumor cells were exposed to 25, 50, and 100 micrograms/ml cisplatinum in medium for varying time periods (4-72 hours). Four changes were consistent: cell rounding and reduced nuclear-cytoplasmic ratio, nuclear chromatin clumping, vesiculation and swelling of the golgi apparatus, and dilatation of the smooth endoplasmic reticulum. These morphologic changes are distinct for cisplatinum and unlike those induced by BCNU (plasma membrane blebbing) and AZQ (mitochondrial swelling and destruction) previously seen in our laboratory. The cellular events described here suggest that cytoplasmic, as well as nuclear, changes (occurring within the same time intervals) may both be relevant to the antitumor effects of cisplatinum.

Effect of phorbol esters on the susceptibility of a glioma cell line to lymphokine-activated killer cell activity.

The mechanisms by which lymphokine-activated killer (LAK) cells exert their cytotoxic effects are not well understood. This study demonstrates that phorbol ester pretreatment of a LAK cell-sensitive glioma cell line (SNB-19) induced a significant decrease in the susceptibility of cells to LAK cell-mediated lysis. This effect was produced by low concentrations of the tumor-promoting phorbol ester, phorbol-12,13-myristate acetate (PMA), and was reversible. Protein kinase C (PKC) inhibitors failed to block this phenomenon. No apparent alteration in the ability of LAK cells to bind to their targets was observed. Thus, PMA may have exerted its effects by a mechanism that does not require PKC, or these glioma cells may possess an isozyme of PKC which is insensitive to the inhibitors used in these studies.

Basic fibroblast growth factor-like activity and receptors are expressed in a human glioma cell line.

Basic fibroblast growth factor (bFGF), a potent mitogen and angiogenic peptide, has been examined as an autocrine regulator of glioma cell growth. The addition of purified bovine pituitary bFGF to an established human glioma cell line, SNB-19, doubled the density of these cells in chemically defined medium. Half-maximal stimulation occurred at 8.2 ng/ml (480 pM). Also, human recombinant bFGF (hr-bFGF) significantly enhanced the growth of SNB-19 cells in soft agar. SNB-19 cells expressed both high and low affinity binding sites for hr-bFGF. These cells expressed approximately 13,000 high affinity sites/cell (Kd = 16.6 +/- 1.7 pM) and 9.5 x 10(6) low affinity sites/cell (Kd = 61.2 +/- 4.1 nM). The results of cross-linking experiments with iodinated hr-bFGF demonstrated the presence of two bands with molecular masses of 145 and 130 kDa. High affinity receptors were also demonstrated in SNB-19 tumors grown in nude mice. SNB-19 cell extracts contained mitogenic activity that eluted from heparin-agarose with high salt (1.2-2 M NaCl) and exhibited many properties normally associated with authentic bFGF. This material cross-reacted with a monoclonal antibody to hr-bFGF, comigrated with hr-bFGF by Western blot analysis, competed with 125I-hr-bFGF in a radioreceptor assay, and stimulated SNB-19 cell growth. These results indicate that a human glioma cell line both expresses and utilizes a bFGF-like growth factor. Such a factor may be an important autocrine regulator of glioma cell growth and may also facilitate its neoplastic progression.

Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate.

The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.