Effects of dicyclohexylamine sulfate, a spermidine synthase inhibitor, in 9L rat brain tumor cells.

Growth characteristics, polyamine levels, and distribution of cells in the cell cycle were determined for 9L rat brain tumor cells treated for various periods with 1 mM dicyclohexylamine sulfate (DCHA). Continuous treatment of cells with DCHA caused growth inhibition at 2 days of treatment. After 2 days of treatment the growth rate of cells increased to approximately the same rate as control cells, even though treatment was continuous. Levels of spermidine were depleted to less than 10% of control levels, spermine levels were essentially unchanged, and putrescine levels were elevated to more than 350% of control levels after 9L cells were treated with DCHA for 2 days. In contrast to results found for the polyamine biosynthesis inhibitor alpha-difluoromethylornithine, treatment of 9L cells with DCHA did not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea. To mimic the effects on polyamine levels caused by treatment with DCHA, 9L cells were treated with 5 mM putrescine alone or with 5 mM putrescine and 1 mM DCHA after treatment with 1 mM alpha-difluoromethylornithine. Results of these experiments suggest that treatment with DCHA alone does not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea because elevated levels of putrescine caused by treatment counteract the effects of decreased spermidine levels.

alpha-Difluoromethylornithine alters calcium signaling in platelet-derived growth factor-stimulated A172 brain tumor cells in culture.

alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h but less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.

ERBB-2 (HER2/neu) gene copy number, p185HER-2 overexpression, and intratumor heterogeneity in human breast cancer.

Amplification of the ERBB-2 (HER-2/neu) gene is accompanied by overexpression of its cell surface receptor product, p185HER-2. Heterogeneity has been observed for both the gene copy number and the level of overexpression of its protein product. To better understand their relationship, correlation between the level of cellular expression of p185HER-2 and ERBB-2 gene amplification was studied in four human breast cancer cell lines (BT-474, SK-BR-3, MDA-453, and MCF-7) and in a primary human breast tumor sample. The relative expression of p185HER-2 was measured by immunofluorescence by using flow and/or image cytometry while correlated DNA analysis was performed on the same cells by fluorescence in situ hybridization to determine ERBB-2 gene and chromosome 17 copy numbers. Marked heterogeneity was observed in both protein expression and ERBB-2 copy number. Despite this heterogeneity, and in accordance with previous studies, the average levels of p185HER-2 expression correlated well with average ERBB-2 gene copy numbers in the four lines examined (r = 0.99). When the relationship between copy number and protein expression was studied on a cell-by-cell basis, p185HER-2 expression correlated with both the absolute number of ERBB-2 gene copies/cell (r = 0.59-0.63) and chromosome 17 copy number (r = 0.45-0.61). It is of interest that there was weak or no correlation between p185HER-2 protein expression and the ERBB-2 copy number:chromosome 17 copy number ratio (r = 0.0-0.25). In more than one-half of cells expressing a high level of p185HER-2, the chromosome 17 copy number was high (two or three times the average copy number), whereas < 2% of an unselected population had a high chromosome 17 copy number. Bromodeoxyuridine incorporation indicated that the S-phase-labeling index was homogeneous across various p185HER-2-expressing subpopulations in the SK-BR-3 cell line. Analysis of the primary breast tumor sample showed results similar to the cell lines, supporting the strong possibility of a mechanistic link among p185HER-2 overexpression, ERBB-2 amplification, and high chromosome 17 copy number.

Effect of N1,N14-bis(ethyl)homospermine on the growth of U-87 MG and SF-126 human brain tumor cells.

The effect of the spermine analogue N1,N14-bis(ethyl)homospermine on the growth, polyamine levels, and survival of U-87 MG and SF-126 human brain tumor cells was examined in tissue culture. At concentrations of 10 mumols and above, N1,N14-bis(ethyl)homospermine inhibited growth significantly, caused a marked decrease in intracellular levels of the naturally occurring polyamines putrescine, spermidine, and spermine, and had a considerable cytotoxic effect on both cell lines after more than 96 h of treatment. In earlier studies we showed that the affinity of the analogue for calf thymus DNA was higher than the affinity of spermine, but that it did not aggregate DNA or release bound ethidium bromide from DNA as efficiently as spermine does. Therefore, the growth-inhibitory and cytotoxic effects of N1,N14-bis(ethyl)homospermine support our hypothesis that polyamine analogues that can enter cells, deplete intracellular levels of natural polyamines, and replace the natural polyamines from their binding sites on DNA without replacing function should act as antiproliferative agents.

Genetic subgroups of anaplastic astrocytomas correlate with patient age and survival.

Astrocytomas are brain tumors with variable responses to radiation and chemotherapy. Tumor grade and patient age are important prognostic factors but do not account for the variability in clinical outcome. We hypothesized that genetic subgroups play a role in the outcome of grade III astrocytomas and studied 80 grade III astrocytomas by comparative genomic hybridization. Some chromosomal aberrations (+7p/q, -9p, -10q, -13q, +19q) were related to aberrations that are frequent in grade IV astrocytoma, whereas others (+10p, -11q, +11p, -Xq) were more frequent in grade III astrocytoma. +7p, +19 and -4q were more frequent in tumors from older patients while -11p was more frequent in tumors from younger patients. Finally, gains of 7p and 7q were associated with shorter patient survival, independent of age. Our results indicate that genetic events underlie the well-known effects of age on survival in grade III astrocytoma and demonstrate the importance of molecular classification in astrocytic tumors.

Effect of 1,19-bis(ethylamino)-5,10,15-triazanonadecane on human tumor xenografts.

The polyamine analogue 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), 5 mg/kg i.p., was given twice daily on days 0-3 and 7-10 (cycle 1) to nude mice with human malignant gliomas (SF-767 and U-87 MG), lung adenocarcinoma (A549), and colon carcinomas (HCT116 and HT29). A second cycle of drug was given to mice with SF-767 and A549 tumors on days 42-45 and 49-52. The maximum animal weight loss varied between 4 and 12%, which was observed 10-15 days following the initiation of treatment, but no overt toxic reactions were noted. The SF-767 brain tumors were extremely responsive to BE-4-4-4-4 alone (3 of 8 complete regressions after 2 cycles); however, the growth of the U-87 MG brain tumor was only slightly inhibited by BE-4-4-4-4 treatment. There was significant inhibition of tumor growth after treatment with one cycle of BE-4-4-4-4 in animals carrying the A549, HCT116, and HT29 tumors. At day 73, the growth of the A549 tumor was inhibited by 78 and 89% following one or two cycles of BE-4-4-4-4, respectively. The mitotic index of A549 tumors was 18 times greater in control mice than in those treated with BE-4-4-4-4 for one or two cycles 99 days after initiation of treatment. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was given to mice carrying the U-87 MG or A549 tumors on day 4 (cycle 1) and day 46 (cycle 2) in the maximal tolerated dose of 50 mg/kg for BCNU alone and 40 mg/kg for BCNU plus BE-4-4-4-4. BCNU alone significantly inhibited the growth of U-87 MG tumors but not the growth of A549 tumors. Treatment with the combination of BCNU and BE-4-4-4-4 was significantly better than BCNU alone for A549 tumors and better than BE-4-4-4-4 alone for U87 tumors. However, in both animal groups treated with the combination, there was a significant weight loss, which was not observed for animals treated with either agent alone. These data suggest a role for BE-4-4-4-4 in the treatment of brain, lung, and colon tumors.

Correlation between the effects of polyamine analogues on DNA conformation and cell growth.

Effects of a number of synthetic analogues of the natural polyamines on the B-Z transition of poly(dG-me5dC) and on the aggregation of calf thymus DNA in solution were studied using circular dichroic and UV spectroscopy. The efficiency of induction of the B-Z transition decreased with a decrease in the length of the central alkyl chain of the analogues, and the ability of analogues to aggregate DNA was markedly reduced for compounds ethylated at the terminal amines. Both structural variations appear to have important effects on the biological functions of polyamines. Most analogues studied depleted intracellular levels of natural polyamines, but only those that did not readily induce the B-Z transition and/or aggregate DNA were good inhibitors of cell growth. All but one of the analogues studied were able to rescue cells--at least in part--from the growth-inhibitory effects of alpha-difluoromethylornithine. The single analogue that was unable to effect rescue also failed to induce both the B-Z transition and the aggregation of DNA.

Interaction of a polyamine analogue, 1,19-bis-(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), with DNA and effect on growth, survival, and polyamine levels in seven human brain tumor cell lines.

Computer graphics modeling and physicochemical studies of spermine-DNA interactions, as well as experiments in cell culture, indicate that a polyamine analogue with strong affinity for nucleic acids but poor ability to condense and aggregate DNA in vitro should act as an antiproliferative agent if it can enter cells. On the basis of our studies of polyamine-DNA interactions, we designed a pentamine, 1,19-bis(ethylamino)-5,10,15- triazanonadecane (BE-4-4-4-4), that had these characteristics. Measurement of melting temperature and ultraviolet light scattering studies show that the affinity of this analogue for calf-thymus DNA is about 4 times higher than that of spermine, whereas its ability to aggregate DNA is slightly poorer than that of spermine. Studies in U-87 MG, U-251 MG, SF-126, SF-188, SF-763, SF-767, and DAOY human brain tumor cells in tissue culture showed that treatment for more than 96 h with concentrations of 5 microM BE-4-4-4-4 or greater inhibited growth; decreased levels of putrescine, spermidine, and spermine; and decreased colony-forming ability in all cell lines. The cytotoxicity of the analogue varied among cell lines; DAOY and SF-767 were the most sensitive and the most resistant lines, respectively. In SF-763 cells, growth inhibition by BE-4-4-4-4 could be partially reversed by the addition of putrescine, spermidine, or spermine 1 day after BE-4-4-4-4 addition, but in U-251 MG cells, growth inhibition was reversed only by spermine and not by other polyamines. When any of the naturally occurring polyamines was added simultaneously with BE-4-4-4-4, growth inhibition was completely blocked. The data suggest that a threshold intracellular concentration of BE-4-4-4-4 is needed to manifest the growth-inhibitory and cytotoxic effects. In most cell lines, once that threshold level is reached, the growth-inhibitory and cytotoxic properties of the analogue are manifest irrespective of cellular polyamine levels. Further increases in the BE-4-4-4-4 concentration or incubation time reduce the intracellular polyamine levels but do not significantly increase growth inhibition. In U-87 MG and DAOY cells, however, prolonged incubation with higher concentrations of BE-4-4-4-4 causes additional growth inhibition along with depletion of intracellular polyamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea by alpha-difluoromethylornithine in chloroethylnitrosourea-sensitive and -resistant 9L rat brain tumor cells in vitro.

alpha-Difluoromethylornithine, an enzyme-activated, irreversible inhibitor of ornithine decarboxylase, inhibited the growth of both chloroethylnitrosourea-sensitive and -resistant 9L rat brain tumor cells in vitro. After 48 hr of treatment with 10 mM alpha-difluoromethylornithine, the putrescine and spermidine contents of both resistant and sensitive cells were less than 5% of control levels, but the spermine level was slightly elevated. The cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea, as measured by a colony-forming efficiency assay, was significantly increased in alpha-difluoromethylornithine-pretreated sensitive cells but not in resistant cells treated with this polyamine inhibitor. With the sister chromatid exchange assay, we found that alpha-difluoromethylornithine pretreatment increased 1,3-bis(2-chloroethyl)-1-nitrosourea-induced damage to chromosomes in sensitive but not in resistant cells.

Design and testing of novel cytotoxic polyamine analogues.

We designed three polyamine analogues, 1,14-diamino-N5-methyl-5,10- diazatetradecane (5me-4-4-4), 1,14-diamino-N5,N5-dimethyl-5,10-diazatetradecane (Q-Amm-4-4-4), and 1,14-bis-(ethylamino)-N5,N5-dimethyl-5,10-diazatetradecane (BE-Q-Amm-4-4-4), on the basis of computer modeling and physical-chemical studies of polyamine-DNA interactions. These analogues differ from natural polyamines and from one another in the charge distribution on their aliphatic backbone. We found that 10 microM 5me-4-4-4 did not inhibit growth and was not cytotoxic to the human brain tumor cell lines SF-767 and SF-126. The same concentrations of Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 inhibited cell growth and killed more than 90% of each cell type on day 7 of the experiment. BE-Q-Amm-4-4-4 was slightly more toxic than Q-Amm-4-4-4 in both cell lines. All three agents either decreased or completely depleted intracellular putrescine and spermidine. Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 each also lowered spermine. The fact that 5me-4-4-4 was nontoxic but that Q-Amm-4-4-4 was cytotoxic and inhibited growth suggests that the charge distribution along the surface of the aliphatic backbone of polyamines is important in determining growth inhibition and cytotoxicity.

Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype.

Allelic alterations of chromosomes 1 and 19 are frequent events in human diffuse gliomas and have recently proven to be strong predictors of chemotherapeutic response and prolonged survival in oligodendrogliomas (Cairncross et al., 1998; Smith et al., submitted). Using 115 human diffuse gliomas, we localized regions of common allelic loss on chromosomes 1 and 19 and assessed the association of these deletion intervals with glioma histological subtypes. Further, we evaluated the capacity of multiple modalities to detect these alterations, including loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The correlation coefficients for detection of 1p and 19q alterations, respectively, between modalities were: 0.98 and 0.87 for LOH and FISH, 0.79 and 0.60 for LOH and CGH, and 0.79 and 0.53 for FISH and CGH. Minimal deletion regions were defined on 19q13.3 (D19S412-D19S596) and 1p (D1S468-D1S1612). Loss of the 1p36 region was found in 18% of astrocytomas (10/55) and in 73% (24/33) of oligodendrogliomas (P < 0.0001), and loss of the 19q13.3 region was found in 38% (21/55) of astrocytomas and 73% (24/33) of oligodendrogliomas (P = 0.0017). Loss of both regions was found in 11% (6/55) of astrocytomas and in 64% (21/33) of oligodendrogliomas (P < 0.0001). All gliomas with LOH on either 1p or 19q demonstrated loss of the corresponding FISH probe, 1p36 or 19q13.3, suggesting not only locations of putative tumor suppressor genes, but also a simple assay for assessment of 1p and 19q alterations as diagnostic and prognostic markers.

1H and 31P nuclear magnetic resonance studies of spermine binding to the Z-DNA form of d(m5CGm5CGm5CG)2. Evidence for decreased spermine mobility.

The binding of spermine to the d(m5CGm5CGm5CG) duplex has been studied by proton and phosphorus nuclear magnetic resonance techniques in order to investigate the mobility and nature of spermine bound to the resulting Z-DNA complex. A characterization of the B to Z transition as a function of increasing spermine concentration demonstrated doubling of the non-exchangeable proton and the phosphorus peaks at a ratio of about 1:1 (spermine/duplex) and a re-simplification of the spectrum at 2:1 (spermine/duplex) where about 90% or the DNA was fully converted into the Z-form. However, some of the Z-DNA proton chemical shifts differed between the 1:1 and 2:1 titration points. Since these differences involved primarily the exchangeable terminal imino and amino protons, they could result from end effects. Discrepancies were generally not observed with the non-terminal proton shifts nor with the phosphorus shifts. These proton and phosphorus chemical shift changes are fully consistent with a B to Z transition. Complexed spermine peaks appear about 0.1 parts per million upfield from the uncomplexed form. The spermine and both the B and Z-DNA hexamer signals are noticeably broadened at the 1:1 ratio but the remaining signals re-sharpen at the 2:1 ratio. Both one-dimensional and two-dimensional studies revealed negative nuclear Overhauser effect (NOE) contacts between each spermine proton. Therefore, spermine has a longer correlation time than that observed for unbounded spermine. These results are contrasted with the positive NOE contacts observed for the B-DNA-spermine complexes reported by Wemmer et al. using the dodecamer d(CGCGAATTCGCG)2 and reported here using the hexamer d(ATGCAT)2. While the mobility of spermine in the Z-DNA complex is significantly less than that of the B-DNA complex, no clear evidence of intermolecular spermine-DNA proton NOE contacts is observed.

Cytoplasmic microinjection of immunoglobulin Gs recognizing RNA helices inhibits human cell growth.

We report here that nucleolar and cytoplasmic RNA in mammalian cells is recognized specifically by both experimentally induced monoclonal IgG unique for left-handed Z-RNA and by autoimmune mouse monoclonal IgG specific for ribosomal RNA. Nucleolar Z-RNA synthesis, like nucleolar ribosomal RNA synthesis, is inhibited by actinomycin D treatment and dimethylsulfoxide-induced differentiation. Immune anti-Z-RNA IgGs microinjected into living nuclei bind nucleolar RNA, and these complexes appear to be removed from the nucleus within minutes. Cytoplasmically microinjected monoclonal or polyclonal anti-Z-RNA IgGs specifically bind cytoplasmic RNA and inhibit cell multiplication. Microinjection of antibodies directed against double-stranded RNAs. Elevated ionic conditions, which in energy-minimized models can cause the walls of the groove in Z-RNA (but not Z-DNA) to approach each other and close, also prevent antibody binding to specific synthetic or cellular Z-RNA determinants. Our antibodies binding unique Z-RNA structures probably recognize antigens determined by the exposed 2'-OH ribose sugar-phosphate groups.

Chromosomal abnormalities in glioblastoma multiforme by comparative genomic hybridization: correlation with radiation treatment outcome.

Glioblastoma multiforme (GM) is the most common and most malignant astrocytoma in adults. After surgery, radiation therapy extends patient survival; however, in vivo response to radiation therapy is variable. The purpose of this investigation was to determine whether the cytogenetic abnormalities of GM differ according to patient response to radiation therapy. Radiation response was defined by either progression [radiation-resistant (RR)] or resolution [radiation-sensitive (RS)] of tumor at the first postradiation radiographic imaging evaluation. Twenty RR and 10 RS frozen tissue specimens were subjected to cytogenetic analysis by comparative genomic hybridization. RS and RR specimens had different cytogenetic aberrations that mapped predominantly to chromosomes 7, 9, 10, 13, and 19. Relative gain of 7 occurred in 70% of the RR and 30% of the RS cases and was the most significant difference involving a single change between the two groups (P = 0.06). RR and RS specimens also differed in their patterns of simultaneous cytogenetic aberrations. A simultaneous gain of chromosomes 7 and 19 was found in 30% of the RR cases but was absent in the RS group. Concurrent loss of 9p23-24 and 13q14 regions was absent in the RS cohort but occurred in 30% of the RR series. This latter cytogenetic pattern was also associated with older age. Amplifications were more common in the RR series, but the difference did not reach statistical significance. The data suggest that GM with different in vivo responses to radiation therapy also differ cytogenetically.

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

Detection of p16 gene deletions in gliomas: a comparison of fluorescence in situ hybridization (FISH) versus quantitative PCR.

The p16 protein plays a key role in cell cycle control by preventing CDK4 from inactivating the retinoblastoma protein (pRb). The corresponding tumor suppressor gene (p16/MTS1/CDKN2) has recently been implicated in malignant progression of astrocytomas and could potentially serve as an important marker for patient prognosis and for guiding specific therapeutic strategies. We have undertaken a study to evaluate 2 methods of detecting p16 deletion. Thirty diffuse gliomas were analyzed for p16 gene dosage. Dual color fluorescence in situ hybridization (FISH) was performed on cytologic preparations using paired centromeric (CEN) and locus-specific probes for CEN9/p16, CEN8/RB, and CEN12/CDK4. Quantitative PCR was performed using primers for p16, MTAP, and reference genes. Eleven cases were also studied using comparative genomic hybridization (CGH). Abnormalities of the p16-CDK4-RB pathway were identified in 21 (70%) cases by FISH and/or PCR. These included 15 (50%) with p16 deletion, 9 of which were detected by both techniques, 3 by FISH alone, and 3 by PCR alone (concordance rate = 81%). FISH analysis further revealed tetraploidy/aneuploidy in 14 (47%), RB deletion in 11 (37%) and CDK4 amplification in 1 (3.3%). There were 94% and 100% concordance rates between CGH and FISH or PCR, respectively. Quantitative PCR was noninformative in 4 cases. Although FISH and quantitative PCR are both reliable techniques, each has limitations. PCR is likely to miss p16 deletions when there is significant normal cell contamination or clonal heterogeneity, whereas the p16 YAC probe used for FISH analysis may miss small deletions. Replacement of the latter with a cosmid probe may improve the sensitivity of FISH in future experiments.

Astroblastoma: clinicopathologic features and chromosomal abnormalities defined by comparative genomic hybridization.

Astroblastomas are uncommon brain tumors whose classification and histogenesis have been debated. Precise criteria for diagnosis have been described only recently, but have not found wide acceptance. We report the clinical, radiographic, and histopathologic features of 20 astroblastomas, and the chromosomal alterations in seven cases as detected by comparative genomic hybridization (CGH). The tumors occurred both in children and young adults (average age, 14 years), most often as well circumscribed, peripheral, cerebral hemispheric masses. Radiographically, the lesions were contrast-enhancing and solid, often with a cystic component. All were characterized histologically by astroblastic pseudorosettes, and most displayed prominent perivascular hyalinization, regional hyaline changes, and pushing borders in regard to adjacent brain. Tumor cells were strongly immunoreactive for S-100 protein, GFAP, and vimentin. Staining for EMA was focal. Ten of 20 astroblastomas were classified as "well differentiated" and 10 were classified as "malignant," largely on the basis of hypercellular zones with increased mitotic indices, vascular proliferation, and necrosis with pseudopalisading. All 10 well differentiated lesions and 8 of 10 malignant lesions were completely resected. None of the well differentiated astroblastomas recurred within the limited follow-up period. Three malignant astroblastomas recurred, including two incompletely resected tumors, and one that had been totally resected. One patient died of disease following recurrence. The most frequent chromosomal alterations detected by CGH were gains of chromosome arm 20q (4/7 tumors) and chromosome 19 (3/7). The combination of these gains occurred in three, including two well differentiated and one malignant astroblastoma. Other alterations noted in two tumors each were losses on 9q, 10, and X. These chromosomal alterations are not typical of ependymoma or infiltrating astrocytic neoplasms, and suggest that astroblastomas may have a characteristic cytogenetic profile in addition to their distinctive clinical, radiographic, and histopathologic features.

Effects of ciglitazone on blood pressure and intracellular calcium metabolism.

Ciglitazone is the prototype of the thiazolidinedione class of compounds currently being developed for the treatment of insulin resistance and non-insulin-dependent diabetes. The effects of thiazolidinediones on blood pressure and cell calcium metabolism are not well defined. In the obese Zucker rat, a widely studied model of insulin resistance associated with mild hypertension, we investigated the effects of ciglitazone on plasma insulin levels and mean arterial pressure. We also evaluated the effects of ciglitazone on the changes in cytosolic calcium induced by platelet-derived growth factor in A172 human glioblastoma cells and rat A10 vascular smooth muscle cells. Oral administration of ciglitazone, approximately 45 mg/kg per day for 4 weeks, induced significant reductions in plasma insulin levels (p < 0.001) and blood pressure (p < 0.05). Ciglitazone was also found to significantly attenuate the capacity of platelet-derived growth factor BB homodimer to induce sustained increases in intracellular free calcium. These findings suggest that thiazolidinediones may offer a novel pharmacological approach to the treatment of hypertension, and raise the possibility that these compounds may affect blood pressure not only by affecting insulin metabolism but also by modifying the cell calcium response to pressor agents, growth factors, or both.

Tissue microdissection and degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) is an effective method to analyze genetic aberrations in invasive tumors.

We amplified various amounts of DNA derived from frozen SF210 and U251NCI human glioblastoma cells, carried out comparative genomic hybridization (CGH) using degenerate oligonucleotide primed-PCR (DOP-PCR) products as test probes, and compared results to analyses performed with probes prepared by standard nick translation. Next we extracted DNA from hematoxylin-eosin (HE)- and methyl green (MG)-stained, microdissected sections of formalin-fixed and paraffin-embedded U251NCI cells, amplified and labeled it by DOP-PCR, and subjected it to CGH. Finally, we used the same methods in multiple samples from a single human mixed glioma tissue. DOP-PCR products from 50 pg to 250 ng of DNA were equally effective in generating the same CGH profiles as the standard method. DOP-PCR products from microdissected pieces of MG-stained cells were effective probes for CGH, but HE-stained samples were not desirable. As the proportion of HE-stained sample increased, CGH profiles deteriorated. DOP-PCR products from microdissected pieces of MG-stained paraffin sections of glioma tissue produced CGH profiles compatible with their histological features. CGH performed with DOP-PCR products from microdissected paraffin blocks allows for the accurate investigation of the cytogenetic characteristics from invasive tumors and of cytogenetic heterogeneity within neoplastic tissue.

Detection of p16, RB, CDK4, and p53 gene deletion and amplification by fluorescence in situ hybridization in 96 gliomas.

Inactivation of the p53 gene is a common early event of astrocytoma tumorigenesis. Alternatively, since the p16, retinoblastoma (RB), and CDK4 genes have been implicated in malignant progression, detection of losses or amplifications of these genes in gliomas could be diagnostically, prognostically, and therapeutically important. We obtained smear preparations from 96 diffuse gliomas and 10 nonneoplastic specimens. Dual-color fluorescence in situ hybridizations using paired probes for CEN9/p16, CEN8/RB, CEN17/p53, and CEN12/CDK4 were performed and revealed expected frequencies of abnormalities, except for p53 losses, which were low (7%). The latter supports the concept that p53 inactivation usually occurs by mitotic recombination. Detected abnormalities of the p16/RB/CDK4 pathway were highly associated with astrocytic differentiation and were univariately associated with decreased patient survival. However, only patient age and histologic classification retained statistical significance on multivariate analysis. We conclude that in diffuse gliomas, p16/RB/CDK4 abnormalities are markers of astrocytic phenotype. Thus, their detection by fluorescence in situ hybridization may have diagnostic usefulness in cases with equivocal morphologic features. Although our numbers are small, we find no additional prognostic significance to these genetic abnormalities one age, grade, and oligodendroglial histology are taken into account.