Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer's disease activates expression of proapoptotic markers in primary cortical neurons.

Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.

Microleakage evaluation of restorations prepared with air abrasion.

PURPOSE: The objective of this in-vitro study was to measure and compare microleakage around preventive resin restorations prepared conventionally or with air abrasion, in the presence or absence of acid etching. METHODS: One hundred extracted human non carious molars were assigned to each of four groups. Group A: fissures opened with a high speed handpiece, etched, and restored with composite and sealant. Group B: fissures opened with KCP 1000 and restored with composite and sealant. Group C: fissures opened with KCP 1000, etched, and restored with composite and sealant. No adhesive was used for groups A, B, or C. Group D: fissures opened with KCP 1000, adhesive resin was applied, cured, and restored with composite and sealant. All teeth were thermocycled, stained with silver nitrate, sectioned and viewed with a computer linked measuring microscope. Measurements were recorded in relative percentages and absolute millimeters. RESULTS: One-way ANOVA and two sample independent t-test showed no statistical significance between groups A and C, or between groups B and D. Statistically significant differences were found among groups A and B, groups A and D, groups B and C, and groups C and D (P < 0.0001). CONCLUSION: The use of air abrasion alone does not provide adequate sealability of preventive restorations.

A possible role for cysteine proteinase and its inhibitors in motility of malignant melanoma and other tumour cells.

The metastasis of malignant tumour cells depends on their rapid replication, and their ability to adhere to the matrix of a biological barrier such as basement membrane, to degrade the matrix, and to migrate through this more permeable barrier. Secreted enzymes, including the cysteine proteinases cathepsins B and L, are known to degrade basement membrane components. Using a barrier-free substratum we studied the possible role of cysteine proteinases in influencing the motility per se of metastatic cells. We found that stefins, the natural inhibitors of cysteine proteinases, markedly decreased the stimulated motility of both human melanoma cells and W256 carcinosarcoma cells at low concentrations (0.5 microM). A stefin also inhibited melanoma cell adherence, but to a lesser extent than motility. Additionally, synthetic inhibitors (E-64, diazomethyl ketones) of cysteine proteinases were found to depress stimulated motility of W256 cells. These results suggest that cysteine proteinases and their inhibitors may have a direct role in the development of a migratory response per se in tumour cells.

Tumor collagenase-stimulating factor and tumor autocrine motility factor as tumor markers in bladder cancer--an update.

We have studied the role of combined measurements of tumor collagenase-stimulating factor (TCSF) and tumor autocrine motility factor (TAMF) in 83 first morning voids and 24-hour specimens in patients with bladder and renal cancer and control subjects. TCSF and TAMF were measured by immunoabsorption assay and motility test utilizing modified Boyden chamber. The mean concentrations of urinary TCSF and TAMF from cancer patients (n = 32) were 4- to 5-fold higher than those from benign conditions (n = 70). We compared TCSF and TAMF utilizing motility test in first morning voids (n = 18) and 24-hour urinary samples (n = 19). TCSF and TAMF were almost identical in terms of eluted proteins from morning voids and 24-hour urine samples. Invasive bladder cancer (n = 12) showed a significantly higher correlation (r = 0.9 and p = 0.0001) between motility response and urinary concentration of TCSF and TAMF. We have also localized these two glycoproteins in cell membranes and cytoplasms of cancer cell.

A new method for evaluation of urinary autocrine motility factor and tumor cell collagenase stimulating factor as markers for urinary tract cancers.

Over the past several years, many tumor markers, including cell surface antigens, T-antigen, ras p55, and ras p52 proteins, have been studied as potential tumor markers of bladder cancer. The lack of specificity and inconsistency of these markers led us to develop a new method for studying the urinary excretion of autocrine motility factor (uAMF) and tumor cell collagenase stimulating factor (TCSF) in 24-hour and first morning voided specimens. AMF is a glycoprotein secreted by the malignant cells and is responsible for cell locomotion, a key event in invasion and metastases of the malignant cells. TCSF is a membrane bound glycoprotein of tumor cells that stimulates fibroblast collagenase production. We have utilized an enzyme-linked immunoabsorption assay to detect the levels of uAMF and TCSF in urine samples collected from normal volunteers, patients with benign diseases, and patients with bladder cancer. Our data indicate that urinary concentrations of uAMF and TCSF are elevated in patients with bladder cancer. Furthermore, the levels of uAMF and TCSF are more elevated in invasive tumors as compared with benign counterparts. We have localized uAMF and TCSF in bladder cancer cells, utilizing immunohistologic techniques.

Anti-laminin receptor antibody targeting of liposomes with encapsulated doxorubicin to human breast cancer cells in vitro.

The tumor cell laminin receptor is a cell-surface protein that binds laminin with high affinity (Kd = 1.0 nM). The putative ligand-binding domain of the laminin receptor has been molecularly cloned and sequenced. In the present study, we used the predicted amino acid sequence of the laminin receptor to generate synthetic peptide antigens and produced immunoglobulin M (IgM) anti-laminin receptor monoclonal antibodies. The disulfide bond group of the IgM molecule was used to couple the antibodies to the surface of liposomes encapsulating doxorubicin. The anti-laminin receptor monoclonal antibodies coupled to the liposomes bound avidly to the surface of MDA-MB-435S (MDA-435) human breast carcinoma cells, which have high numbers of laminin receptors. These antibody-coupled liposomes exhibited a low degree of binding to Hs 578Bst (Hs 578) normal human breast epithelial cells, which express a low number of laminin receptors. Excess liposomes competed for the binding of unbound laminin to the tumor cell surface, and excess laminin competed for binding with the liposomes. Antibody-coupled liposomes encapsulating doxorubicin were specifically more efficient in inhibiting colony formation by MDA-435 cells in vitro than unbound doxorubicin or liposomes without anti-laminin receptor monoclonal antibodies. Unbound doxorubicin inhibited thymidine uptake by 10%-20% in both Hs 578 and MDA-435 cells, whereas the antibody-coupled liposomes encapsulating doxorubicin inhibited thymidine uptake by 90% in MDA-435 cells but only 15% in Hs 578 cells. Thus, use of anti-laminin receptor monoclonal antibodies coupled with liposomes encapsulating doxorubicin represents a new strategy for selective targeting of doxorubicin to carcinoma cells with exposed laminin receptors.

Detection of autocrine motility factor in urine as a marker of bladder cancer.

Cell locomotion is an essential requirement for invasion and metastasis of malignant cells. We have previously described the characterization of a 50-kilodalton autocrine motility factor (AMF), a cytokine that stimulates motility in human tumor cells. In this study, we investigated the elaboration of this factor in vivo by human bladder carcinoma and in vitro by a cultured transitional cell carcinoma (TCC) of the bladder cell line T24P. Urine samples from patients with bladder cancer were assayed for their capacity to stimulate migration of tumor cells. Comparing all TCC cases (22 patients) with all nonmalignant diagnoses (27 patients), we found a statistically significant (P less than .001) difference in the motility values. Invasive TCC cases (15 patients) were significantly (P less than .002) higher in regard to motility values compared with noninvasive TCC cases (8 patients), including one case of carcinoma in situ. In follow-up screening studies evaluating TCC recurrence, the recurrent tumors (9 patients) were higher (P less than .001) in regard to motility values than the tumor-free cases (11 patients). Furthermore, T24P cells showed a dose-dependent motile response to their own serum-free conditioned medium as well as to the AMF present in the urine of TCC patients. This finding is consistent with the source of AMF in the urine of these patients being the cancer itself. An enzyme-linked immunosorbent assay (ELISA) for AMF was also developed. Values determined by ELISA correlated well with the motility values measured separately. These data support the potential usefulness of AMF as a urine marker for bladder TCC.

Biochemical mechanisms of tumor invasion and metastases.

Cancer invasion and metastases is a complex multistep process. In order for a tumor cell to successfully traverse all the steps of this process and initiate a metastatic colony, it must express the right combination of gene products. Such gene products may include proteins which regulate cell interaction with the basement membrane and cell motility. Tumor cells attach to the basement membrane glycoprotein laminin via the cell surface laminin receptor. The human laminin receptor was purified and molecularly cloned. The level of laminin receptor mRNA is a variety of human carcinoma cells correlated with the number of laminin receptors on the cell surface of these cells. Following attachment to the basement membrane, the tumor cell next secretes proteases which may degrade type IV collagen. A genetic linkage between type IV collagenase secretion and metastases was studied using our new genetic system for inducing metastases employing the ras oncogene. Following attachment and local proteolysis, the third step of invasion is tumor cell motility. We have isolated a tumor cell autocrine motility factor (AMF). This factor is secreted by the tumor cells and binds to a cell surface receptor resulting in a profound (greater than 100x) stimulation of cell locomotion. AMF may play a major role in the autonomous invasive behavior of tumor cells.

Biochemical mechanisms of tumor invasion and metastases.

Cancer invasion and metastases is a complex multistep process. In order for a tumor cell to successfully traverse all the steps of this process and initiate a metastatic colony, it must express the right combination of gene products. Such gene products may include proteins which regulate cell interaction with the basement membrane and cell motility. Tumor cells attach to the basement membrane glycoprotein laminin via the cell surface laminin receptor. The human laminin receptor was purified and molecularly cloned. The level of laminin receptor mRNA is a variety of human carcinoma cells correlated with the number of laminin receptors on the cell surface of these cells. Following attachment to the basement membrane, the tumor cell next secretes proteases which may degrade type IV collagen. A genetic linkage between type IV collagenase secretion and metastases was studied using our new genetic system for inducing metastases employing the ras oncogene. Following attachment and local proteolysis, the third step of invasion is tumor cell motility. We have isolated a tumor cell autocrine motility factor (AMF). This factor is secreted by the tumor cells and binds to a cell surface receptor resulting in a profound (greater than 100x) stimulation of cell locomotion. AMF may play a major role in the autonomous invasive behavior of tumor cells.

Biochemical mechanisms of tumor invasion and metastasis.

Cancer invasion and metastases is a complex multi-step process. In order for a tumor cell to successfully traverse all the steps of this process and initiate a metastatic colony, it must express the right combination of gene products. Such gene products may include proteins which regulate cell interaction with the basement membrane and cell motility. Tumor cells attach to the basement membrane glycoprotein laminin via the cell surface laminin receptor. The human laminin receptor was purified and molecularly cloned. The level of laminin receptor mRNA in a variety of human carcinoma cells correlated with the number of laminin receptors on the surface of these cells. Following attachment to the basement membrane, the tumor cell next secretes proteases which may degrade type IV collagen. A genetic linkage between type IV collagenase secretion and metastases was collagen. A genetic linkage between type IV collagenase secretion and metastases was studied using our new genetic system for inducing metastases by employing the ras oncogene. Following attachment and local proteolysis, the third step of invasion is tumor cell motility. We have isolated a tumor cell autocrine motility factor (AMF). This factor is secreted by the tumor cells and binds to a cell surface receptor, resulting in a profound (greater than 100 x) stimulation of cell locomotion. AMF may play a major role in the autonomous invasive behavior of tumor cells.

Cytokine-induced pseudopodial protrusion is coupled to tumour cell migration.

Pseudopodia protrusion is a prominent feature of actively motile cells in vitro and invading tumour cells in vivo; however, the function and regulation of pseudopodia are poorly understood. Tumour autocrine motility factor (AMF) represents a new class of cytokines which are secreted by tumour cells and embryonic cells and induce random motility in the producer cells or in heterologous cells with appropriate receptors. Here we report that a major effect of this factor is to induce the extension of cell pseudopodia before cell translocation. Using a new method to quantify and isolate pseudopodia, we find that human breast carcinoma cell AMF (at concentrations of 1 nM or below) stimulates random pseudopodia formation in a dose-dependent and time-dependent manner. Anti-AMF antibodies inhibit pseudopodia protrusion and cell motility, showing the importance of pseudopodia formation during locomotion. AMF-stimulated motility and pseudopodia formation occur on a wide variety of adhesive substrata which suggests that certain intrinsic motility events are independent of the attachment mechanism. Induced pseudopodia show a prominent axial actin network in the electron microscope. The number of laminin receptor and fibronectin RGD recognition sites is increased by a factor of 20 in the induced pseudopodia when compared to the average distribution in unstimulated cells. Exploratory pseudopodia regulated by cell-derived motility factors contain receptors for matrix proteins and could serve as 'senseorgans' essential to the process of cell locomotion.

Pertussis toxin inhibits stimulated motility independently of the adenylate cyclase pathway in human melanoma cells.

The human melanoma cell line, A2058, has previously been shown to respond to an autocrine motility factor (AMF). We have studied biochemical pathways that may be involved in the generation of such a motile response. Pertussis toxin (PT) caused a profound, rapid decrease in stimulated motility that was both dose and time-dependent. Preincubation of cells for 2 hr with as little as 1 ng/ml of PT significantly inhibited motility. A concentration of PT (0.5 microgram/ml) that completely eliminated migration after a 30 min. preincubation had a markedly reduced effect when added 1 hr after the start of the assay. In contrast, agents which selectively modulate or have a role in the adenylate cyclase pathway, e.g., cholera toxin, forskolin, the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate and the cyclase inhibitor 2',5'-dideoxyadenosine, all had negligible effect upon motility. These data are consistent with the presence of a receptor coupled to a PT sensitive G protein initiating motility independently of the adenylate cyclase system.

Studies on the effects of ethinyl estradiol and norethisterone acetate on the adrenal cortex and some other tissues in the rat.

Alkaline phosphatase activity (AP) of the adrenal cortex of rats were determined under the effect of ethinyl estradiol (EE) and/or norethisterone acetate (NA), the two components of the contraceptive pill gyn-anovlar (Schering AG Berlin). A pathological study was also carried out to examine the effects of EE and NA on other tissues mainly the liver, lungs, spleen and ovaries. EE in a dose of 10 micrograms/day for 2 weeks caused a significant increase in the weight of the adrenal but no significant increase in the AP/g cortical tissue. The 25 and 50 micrograms doses for the same period caused a significant increase in both adrenal weight and AP. When treatment was prolonged to 6 weeks no effect on adrenal weight or AP was detected. The same finding was obtained with NA in a dose of 7 mg/rat/day for 2 weeks. The 14 mg dose of NA for the same period caused a significant increase in adrenal weight but no effect on AP. The 21 mg dose caused a significant increase in adrenal weight accompanied by significant decrease in AP/g cortical tissue. Treatment with NA for 6 weeks caused a rise in adrenal weight and AP with the 7 mg dose, then a decline in AP with the 14 mg dose, and a decline in both adrenal weight and AP with the 21 mg dose. As regards the effects of EE and NA on other tissues, EE was found to have a powerful stimulatory effect on the reticuloendothelial system (RES) as well as toxic effects on the liver. NA did not produce such lesions except for the large doses and prolonged periods of treatment. In addition NA produced cholestasis in the liver associated with staining of the liver cells with bile. Combination of EE and NA in the form of gyn-anovlar produced more powerful stimulation of RES and decreased the toxic manifestations of either component. As regards the ovaries, administration of 50 micrograms EE for 6 weeks produced profound hyperplasia of the granulosa cells of the Graafian follicles and inhibited ovulation, however, NA did not inhibit ovulation. With gyn-anovlar, the effect of EE on the ovaries seemed to predominate and ovulation was inhibited.