Transfer of epinastine to infants through human breast milk.

The purpose of this study was to develop an analytical method for analyzing epinastine in breast milk and maternal plasma samples to determine the safety of epinastine in breastfed infants. Six nursing mothers took epinastine hydrochloride (20 mg) once a day for 7 days, while a nursing mother took it for 30 days. Breast milk and blood samples were collected 2, 4, and 10 h after administration from the volunteers. A liquid chromatography-mass spectrometry system was used to analyze samples pretreated by liquid-liquid extractions. The concentration of epinastine in human milk was 10.3-33.5 ng/mL after 2 h, 9.1-63.8 ng/mL after 4 h, and 8.3-28.9 ng/mL after 10 h. The increase achieved 4 h after administration indicates that epinastine was transferred into human breast milk. However, the milk-to-plasma ratio had a wide range (0.82-3.39), while the relative infant dose at 4 h was 0.36-2.49%, which is lower than the safety level of transferability (10%). Moreover, the plasma levels of epinastine in two infants were slightly below the quantification limit. Overall, our results suggested that epinastine can safely be used by nursing mothers without affecting their infants.

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion.

BACKGROUND: Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs. METHODS: Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines. RESULTS: Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. CONCLUSION: Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Visualization of angiographical arteriovenous shunting in perisylvian glioblastomas.

BACKGROUND: Arteriovenous shunting visualized by angiography is one of the major features of glioblastomas, and the visualization is dependent on the presence of extensive shunting. Extensive arteriovenous shunting is associated with the risk of poorly controlled intraoperative bleeding. When a tumor with extensive arteriovenous shunting is located in close proximity to the eloquent regions of the brain, a meticulous surgical procedure is necessary. In the present study, the site-oriented visualization of angiographical arteriovenous shunting was evaluated from the perspective of surgical treatment, with a particular focus on the perisylvian region that is in close proximity to motor and language regions (dominant hemisphere), as well as large arteries and veins. METHODS: Twenty-six consecutive patients underwent a resection of glioblastoma between February 2007 and September 2012. All patients were presurgically examined using digital subtraction angiography. The patients were subdivided into the following two groups based on the location of the tumor: 1) perisylvian glioblastoma (18 patients) and 2) non-perisylvian glioblastoma (eight patients). Angiography to detect the arteriovenous shunting was performed. In addition, the number of intratumoral vessels, tumor proliferative activity (MIB-1 labeling index), and volume of intraoperative bleeding were evaluated and compared between the two groups. RESULTS: Angiographical arteriovenous shunting was definitively visualized in 13 of 18 (72 %) perisylvian glioblastomas, in contrast to only one of eight (13 %) non-perisylvian glioblastomas (p = 0.007). There were no significant differences between the two groups with respect to the number of intratumoral vessels, MIB-1 labeling index, and volume of intraoperative bleeding. However, massive intraoperative bleeding of > 2,000 mL occurred in one perisylvian glioblastoma patient. CONCLUSIONS: Glioblastomas in the perisylvian region tend to be associated with extensive arteriovenous shunting that can be definitively visualized by performing an angiography. Because arteriovenous shunting carries the risk of intraoperative bleeding, perisylvian glioblastomas-particularly in the dominant hemisphere-should be resected with a meticulous surgical procedure and strategy.

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy.

BACKGROUND: Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues. METHODS: To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines. RESULTS: Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells. CONCLUSION: These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.

A unique ectonucleotide pyrophosphohydrolase associated with porcine chondrocyte-derived vesicles.

Previous studies have shown increased nucleotide pyrophosphohydrolase (EC 3.6.1.8) (NTPPHase) activity in detergent extracts of degenerated human cartilage containing calcium pyrophosphate dihydrate (CPPD) crystals relative to those from osteoarthritis or normal cartilage. NTPPHase was later shown to be an ectoenzyme and its activity was increased in synovial fluid from patients with CPPD crystal deposits relative to fluids from other types of arthritis. We have purified a soluble 61-kD NTPPHase from conditioned media of organ-cultured porcine articular cartilage to electrophoretic homogeneity. Its NH2-terminal sequence through 26 cycles showed < 30% homology to any previously reported protein sequence. An antibody raised to a synthetic peptide corresponding to this sequence reacted with denatured but not native enzyme. This antibody reacted against a sedimentable vesicle-associated 127-kD protein in conditioned media from cultured articular cartilage or from chondrocytes in primary monolayer culture and against a series of soluble proteins in conditioned media supernatant, including a 61-kD protein representing our original isolate. No reactivity was found in 1% SDS extracts of washed cultured chondrocytes, although these contained greater NTPPHase activity than the conditioned media. Antibody to PC-1, another ectoNTPPHase, reacted with 1% SDS extracts of whole chondrocytes but not against those chromatographic fractions containing the major portion of NTPPHase activity. Release of the vesicle-associated 127-kD enzyme into conditioned medium was stimulated three- to sevenfold by TGF beta 1. The antibody also reacted with a series of soluble proteins and with 127-kD sedimentable protein in human synovial fluid. Kinetic studies supported the existence of a unique vesicle-associated NTPPHase; apparent Km (mM) of chondrocyte membrane NTPPHase was 1.5 and 3.0 at pH 7.3 and 9.88, respectively; apparent Km (mM) of vesicle associated NTPPHase was 0.83 and 1.28 at pH 7.3 and 9.88. The data suggest the existence of a unique ecto-NTPPHase associated with vesicles derived from normal articular cartilage.

Multiple peripheral middle cerebral artery aneurysms associated with Behcet's disease.

We report a 19-year-old woman with Behcet's disease who suffered a subarachnoid hemorrhage and had bilateral peripheral middle cerebral artery aneurysms. After steroid therapy for 3 days, the smaller aneurysm disappeared. The larger aneurysm was excised and the artery reconstructed using a superficial temporary artery graft. Histological examination showed vasculitis restricted to the wall of the aneurysm. This is the first report of arterial reconstruction for an aneurysm associated with Behcet's disease. Steroid therapy before the operation may facilitate repair of the arterial wall.

Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor: melanoma p75 functions independently of trkA.

Although overexpression of the low-affinity p75 neurotrophin receptor (p75NTR) is frequently associated with advanced stages of human melanoma progression, the functional significance of this finding is unknown. We examined whether the degree of cell surface expression of p75NTR in human melanoma cell variants determines their extent of invasion stimulated by nerve growth factor (NGF). Treatment of MeWo melanoma cells or a metastatic spontaneous wheat germ agglutinin-resistant variant subline (70W) of MeWo cells with 2.5S NGF resulted in a dose-dependent enhancement of invasion through a reconstituted basement membrane. This effect was most pronounced with the 70W subline that exhibits brain-metastasizing potential in nude mice but was not found with a poorly metastatic MeWo variant subline (3S5). The expression of p75NTR as determined by Northern blotting and immunoprecipitation analysis of 125I-labeled cell surface proteins correlated with NGF-stimulated invasion. The MeWo melanoma sublines used in this study did not express p140proto-trkA mRNA or any p140proto-trkA variant transcripts including p70trkA as determined by Northern analysis and RT-PCR analysis. Thus, these melanoma cells would not be expected to form functional p75-p140 heterodimers or p140-p140 homodimers capable of transducing an NGF-generated signal to p140proto-trkA cytoplasmic substrates. These cells did express authentic p145trkC transcripts. However, NGF did not catalytically activate p145trkC receptors via increased tyrosine phosphorylation as would be expected if p145trkC participated in the signaling established by NGF. Furthermore, a NGF-stimulated purine-analogue-sensitive kinase activity was found to coimmunoprecipitate with p75NTR. This p75NTR-associated kinase may coordinate initial signaling events evoked by p75NTR ligand interaction. Addition of 2.5S NGF, at concentrations that should saturate cell surface p75NTR, to matrix-adherent cultures of human MeWo and 70W but not 3S5 melanoma cells suppressed the expression of 92-kDa type IV collagenase and stimulated the production of 72-kDa type IV collagenase in its fully active 68-kDa form. In the absence of p140proto-trkA, the matrix-dependent effects of NGF on metalloproteinase expression of brain-metastatic 70W melanoma cells suggest a signaling role for the low-affinity melanoma p75NTR receptor and its associated purine-analogue-sensitive kinase in signaling enhanced matrix penetration of NGF-rich stromal microenvironments such as the brain.

Clinical evaluation of cellulose porous beads for the therapeutic embolization of meningiomas.

BACKGROUND AND PURPOSE: Cellulose porous beads (CPBs) are a new, exceptionally uniformly sized, nonabsorbable embolic agent. We evaluated their efficacy in the preoperative embolization of meningiomas. METHODS: In 141 consecutive patients, we used CPBs (200-microm diameter) for the preoperative embolization of meningiomas. We selected patients whose tumors were > or =4 cm with 50% of blood to the tumor supplied by the external carotid artery (ECA). All patients underwent a provocation test before embolization. The percentage of blood supplied to the tumor by the internal carotid artery and ECA was determined angiographically. Nonenhanced areas on postembolization MR imaging were calculated. Intraoperative blood loss, units of blood transfusion, and hemostasis at the time of surgery were recorded for each patient. The interval between embolization and surgery was intentionally longer than 7 days. RESULTS: Of the 141 patients, 128 underwent CBP embolization. Eleven patients had positive provocation test results, and 2 had vasospasm; they were not CBP embolized. In 72% of the patients CBP embolization achieved reduction in the flow of the feeding artery by more than 50%. The nonenhanced area on MR imaging was not significantly correlated with the degree of ECA supply or devascularization. The interval between embolization and surgery was 8-26 days (mean, 9.9 days). The longer this interval, the greater was the tumor-softening effect and the rate of tumor removal. CONCLUSIONS: CPBs may be useful for the preoperative embolization of meningiomas. To increase the efficacy of CPB embolization, the interval to surgery should be at least 7 days.

Potential role of calcineurin for brain ischemia and traumatic injury.

Calcineurin belongs to the family of Ca2+/calmodulin-dependent protein phosphatase, protein phosphatase 2B. Calcineurin is the only protein phosphatase which is regulated by a second messenger, Ca2+. Furthermore, calcineurin is highly localized in the central nervous system, especially in those neurons vulnerable to ischemic and traumatic insults. For these reasons, calcineurin is considered to play important roles in neuron-specific functions. Recently, on the basis of the finding that FK506 and cyclosporin A serve as calcineurin-specific inhibitors, this enzyme has become the subject of much study. It is clear that calcineurin is involved in many neuronal (or non-neuronal) functions such as neurotransmitter release, regulation of receptor functions, signal transduction systems, neurite outgrowth, gene expression and neuronal cell death. In this review, we describe the calcineurin functions, functions of the substrates, and the pathogenesis of traumatic and ischemic insults, and we discuss the potential role of calcineurin. There are many similarities in traumatic and ischemic pathogenesis of the brain in which the release of excessive glutamate is followed by an intracellular Ca2+ increase. However, the intracellular cascade which leads to neuronal cell death after the release of excess Ca2+ is unclear. Although calcineurin is thought to be a key toxic enzyme on the basis of studies using immunosuppressants (FK506 or cyclosporin A), many of the functions of the substrates for calcineurin protect against neuronal cell death. We concluded that calcineurin is a bi-directional enzyme for neuronal cell death, having protective and toxic actions, and the balance of the bi-directional effects may be important in ischemic and traumatic pathogenesis.

Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation.

BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.

Metastatic capacity and intercellular communication between normal cells and metastatic cell clones derived from a rat mammary carcinoma.

Three highly metastatic clones and two weakly metastatic clones were obtained from a spontaneously arising mammary carcinoma in an SHR rat. The difference in their capacity to generate metastatic ability was recognized when the tumor cells were implanted s.c. but not when they were implanted i.v. This evidence possibly indicates that the difference in the metastatic capacity of these clones is caused by different potential for detachment from the primary site and for intravasation during the various steps of metastasis. There are no differences between highly and weakly metastatic clones with regard to their in vitro growth characteristics (doubling time, saturation density, plating efficiency, etc.), homotypic aggregation, and adhesiveness to plastic matrices and fibroblast monolayers. Therefore, we used a dye transfer method to examine the relationship between the metastatic capacity of tumor cells and the capacity of tumor cells to make junctional communication with normal fibroblasts. We found that the incidence of intercellular communication between weakly metastatic clone cells and fibroblasts (derived from normal s.c. tissues and tumor tissues) was significantly higher than that between highly metastatic clone cells and fibroblasts. These results suggest that junctional communication between tumor cells and normal fibroblasts may play a part in the early stage of cancer metastasis.

A paracrine migration-stimulating factor for metastatic tumor cells secreted by mouse hepatic sinusoidal endothelial cells: identification as complement component C3b.

Selective malignant cell invasion at secondary sites mediated by organ-specific (paracrine) motility factors may be of importance in preferential organ colonization of metastatic cells. In this study we isolated and examined a migration-stimulating activity present in mouse hepatic sinusoidal endothelial cell-conditioned medium (HSE-CM). HSE-CM contains migration-stimulating activity for highly liver-metastatic (RAW117-H10) and highly lung- and liver-metastatic (RAW117-L17) mouse large cell lymphoma sublines but not for the poorly metastatic parental line (RAW117-P). A migration-stimulating factor for H10 cells was purified from HSE-CM by hydroxylapatite affinity and DEAE anion exchange chromatography, Sephacryl S-200 gel filtration, and preparative native gel electrophoresis. The activity in each of the purification fractions was measured in a Transwell chamber assay using 3-microns diameter pore filters. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the component migrated as a single component of M(r) > 200,000 (nonreducing conditions) or as two components or M(r) approximately 110,000 and approximately 67,000 (reducing conditions). The factor was bound to Concanavalin A-Sepharose but not to heparin- or gelatin-Sepharose affinity columns, induced mainly H10 chemotactic cell activity and some chemokinetic activity, and preferentially stimulated the chemotaxis of liver-colonizing RAW117 sublines (H10 > L17 > P). NH2-terminal amino acid sequence analysis of each subunit indicated that the HSE-CM-derived migration-stimulating factor was a proteolytic fragment of complement component C3. HSE-derived migration-stimulating factors may be important in determining the ability of RAW117 tumor cells to invade and colonize the liver.

Host-mediated therapeutic effects produced by appropriately timed administration of bleomycin on a rat fibrosarcoma.

The timing of bleomycin (BLM) administration after KMT-17 tumor inoculation was found to be important for optimizing its therapeutic effect on tumor-bearing rats. A remarkable therapeutic effect was observed when BLM (5 mg/kg/day) was administered i.p. for 5 days from the eighth day after tumor inoculation (Day 8 to Day 12) rather than when BLM was administered i.p. for 5 days during the days immediately following tumor inoculation (Day 1 and Day 5) (cured rats/treated rats: 10/21 and 2/16, respectively). By means of a Winn assay, stronger tumor-neutralizing activities were observed in spleen cells from BLM (Day 8 to Day 12)-treated tumor-bearing rats than were observed in spleen cells from BLM (Day 1 to Day 5)-treated tumor-bearing rats (% Inhibition: 70.9 and 49.3%, respectively). These therapeutic effects were thus found to be consistent with the antitumor immunity against KMT-17. The enhanced tumor-neutralizing activities of spleen cells from BLM-treated tumor-bearing rats were suppressed by adding spleen cells from nontreated tumor-bearing rats. In cell transfer experiments, an antitumor transplantation resistance in rats immunized with irradiated KMT-17 cells was abrogated by an adoptive transfer of spleen cells from untreated tumor-bearing rats or BLM (Day 1 to Day 5)-treated tumor-bearing rats but not from BLM (Day 8 to Day 12)-treated tumor-bearing rats. These results suggest that, when BLM is administered during a late stage of tumor growth, it is effective in eliminating suppressor cells and that this leads to an improvement in the therapeutic effects of the drug.

Ultrastructural differences in junctional intercellular communication between highly and weakly metastatic clones derived from rat mammary carcinoma.

We examined by electron microscopy the differences in junctional intercellular communications among highly metastatic clones, weakly metastatic clones, and the parent clone obtained from a spontaneously developed rat mammary carcinoma. We also investigated intercellular communications of the highly and weakly metastatic clone cells with normal fibroblasts. The results showed that ultrastructural changes of the highly metastatic clone cells, such as microvilli, microfilaments, and small organelles including endoplasmic reticulum, Golgi apparatus, and mucous particles, were more distinct than those of the weakly metastatic clone cells, and that the numbers of desmosome and gap junctions of weakly metastatic clone cells were significantly greater than those of highly metastatic clone cells. The formation of gap junctions and desmosomes was found only between weakly metastatic clone cells and normal fibroblasts. When both highly and weakly metastatic clone cells were cultured with normal fibroblasts, a tight junction was observed only in the culture of weakly metastatic clone cells and normal fibroblasts. These results suggest that ultrastructural differences are related to the proliferation and detachment of tumor cells from the primary site in the initial stage of tumor metastasis.

Growth-associated shedding of a tumor antigen (CE7) detected by a monoclonal antibody.

A monoclonal antibody was developed against an antigen, termed CE7, which was highly expressed on the surface of rat fibrosarcoma KMT-17 cells (clone A3) cultured in low serum medium (A3-1% FCS). The CE7 antigen was not detectable on A3 cells cultured in ordinary high serum medium (A3-10% FCS), on in vivo passaged A3 cells, or on parental in vivo KMT-17 cell line. However, immunoelectron microscopy and Western blot analyses indicated that CE7 antigen was produced by these tumor cells in all circumstances but was shed from their surfaces in vesicular form into the surrounding tissue culture medium or ascites, unless low serum concentration prevailed and disappeared from their cell surfaces. We have previously reported that the immunogenicity of A3 cells was increased when the serum concentration was lowered from 10% to 1% and the phenomenon paralleled the CE7 antigen expression on the A3 cells. These results suggest that the CE7 antigen could be a tumor-associated rejection antigen and that the expression of the CE7 antigen on A3-1% FCS cells (which is shed by high serum culture or in vivo transplantation and disappears from the cell surface) may play a role in immunological responses against the tumor cells.

Possible participation of tumoricidal macrophages in the therapeutic effect of bleomycin on a transplantable rat fibrosarcoma.

When bleomycin (BLM) (5 mg/kg/day) was administered i.p. to WKA rats for 5 days from the eighth day after KMT-17 implantation, the therapeutic effects of BLM were demonstrated by complete tumor regression in 50% of the cases and prolongation of the mean survival time of the remainder [survival days, 44.3 +/- 13.6 (SD)]. The combined administration of an antimacrophage agent, carrageenan, with BLM significantly inhibited the therapeutic effects of BLM (cure rate, 33%; survival days, 29.8 +/- 5.8). By means of a Winn assay, the tumor neutralizing activity of both spleen cells and peritoneal cells (PC) against KMT-17 cells was found to be augmented in BLM-treated tumor bearing rats as compared with that in nontreated tumor bearing rats. The enhanced tumor neutralizing activity of spleen cells was not abolished by an anti-rat T-cell serum plus complement treatment and was present in an adherent macrophage enriched population. Similarly, an in vitro [125I]iododeoxyuridine release test enabled us to observe the enhancement of the cytotoxic activity of adherent spleen cells and adherent PC in BLM-treated rats. The cytotoxic activity of the PC of BLM-treated rats was not specific to KMT-17 cells alone but was also observed to operate against antigenically different tumor cells such as WFT-2N, KST-20, and K562 cells. An in vitro carrageenan treatment of PC taken from BLM-treated rats reduced their cytotoxic activity. At the same time, the combined administration of carrageenan and BLM also reduced the cytotoxic activity of PC. These results suggest that the tumoricidal activity of macrophages in tumor bearing rats is augmented after BLM therapy and that the activated tumoricidal macrophages may participate in the host-mediated antitumor effects of BLM.

Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme.

Previous experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type p53 protein in a dominant-negative fashion. However, it remains unknown whether tumors with such dominant-negative (transdominant) p53 mutants have a biological significance that is different from that of recessive p53 mutants. In this study, we examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed. We tested a total of 106 p53 mutants, which were identified in brain tumors, glioblastoma multiforme-derived cell lines, breast cancers, or premalignant lesions and squamous cell carcinomas of oral epithelium or were otherwise created by mutagenesis. In agreement with the previous studies, our results demonstrated that transdominant mutations affected amino acid residues that are essential for the stabilization of the DNA-binding surface in the p53 core domain and for the direct interaction of p53 with its DNA-binding sequence. Among 40 patients with sporadic glioblastomas, the average age at diagnosis was significantly younger in the patients with tumors harboring dominant-negative mutations (30.4 +/- 14.7 years, n = 7) than it was in those with recessive mutations (55.2 +/- 18.6 years, n = 9, P < 0.012) and in those without mutations (54.7 +/- 17.1 years, n = 24, P < 0.003). Our data suggest that dominant-negative p53 mutants accelerate development and/or growth of glioblastoma anlagen.

Mechanisms of c-erbB2/neu oncogene-induced metastasis and repression of metastatic properties by adenovirus 5 E1A gene products.

c-erbB2/neu is a transforming oncogene that encodes a 185-kDa transmembrane glycoprotein. In many but not all studies, amplification and/or overexpression of the human c-erbB2/neu oncogene has been correlated with poor prognosis and the number of lymph node metastases in node-positive breast cancer patients. We have shown that expression of the activated rat c-erbB2/neu oncogene in mouse embryo fibroblast 3T3 cells is sufficient to induce experimental metastases in nude mice. Important steps in the metastatic event are tumor cell adhesion to endothelial cells and invasion of basement membranes. Therefore, we further examined the ability of c-erbB2/neu oncogene-transformed 3T3 cells to adhere to microvessel endothelial cells and secrete basement membrane-degradative enzymes. The c-erbB2/neu oncogene-transformed 3T3 cells were shown to be more adherent and have higher gelatinase activities. Since we had previously shown that the adenovirus 5 E1A gene product can suppress c-erbB2/neu-induced transformation of 3T3 cells, we examined the possibility that E1A can abrogate the metastatic properties of c-erbB2/neu-transformed 3T3 cells. We found that introduction of the E1A gene into c-erbB2/neu-transformed 3T3 cells reduced the formation of experimental metastatic tumors and inhibited metastasis-associated properties, such as adhesion to microvessel endothelial cells, migration through a layer of reconstituted basement membrane (Matrigel) and secretion of basement membrane-degradative enzymes. The results indicate that the mechanism by which the c-erbB2/neu gene induces higher metastatic potential is to promote adhesion and invasion steps of the metastatic cascade. The E1A gene, which functions by inhibiting these steps, is thus a suppressor gene for c-erbB2/neu-induced experimental metastasis.