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M Scanlon

Publications and source records attributed to M Scanlon.

At least 37 records · Page 2Linked to original sources
PubMed1993

Reexpression of neu-encoded oncoprotein counteracts the tumor-suppressing but not the metastasis-suppressing function of E1A.

By transfecting the adenovirus 5 E1A gene into neu-transformed NIH3T3 cells, we previously showed that E1A can dramatically repress neu-encoded p185 expression and, concomitantly, suppress the features of transformation and metastasis of neu+E1A transfectants. From these results we concluded that suppression of transformation and metastasis by E1A in neu-transformed cells may be through repression of neu gene expression. However, E1A has recently been shown to also repress the transformation features of other human cancer cells that do not overexpress neu. This observation raised a possibility that repression of neu gene expression in our neu+E1A cells might not be the only mechanism for transformation and metastasis suppression. To study whether other molecular mechanisms might be involved in suppression of transformation and metastasis by E1A in our neu+E1A cells, we reexpressed p185 oncoprotein in the neu+E1A cells by transfecting them with a plasmid containing activated rat neu complementary DNA and we examined whether E1A can suppress transformation and metastasis when the neu-encoded p185 protein is reexpressed. All the features of transformed cells including cell morphology, DNA synthesis rate, colony formation in soft agar, and tumorigenicity in nu/nu mice were restored in the cell lines that reexpressed neu. In addition, the levels of neu reexpression corresponded to the degree of malignant transformation. However, the in vivo metastatic tumor formation by these p185 reexpressing cells was still significantly inhibited by E1A. When metastasis-associated properties were examined in the cell lines that reexpressed p185, we found that cell motility was recovered by reexpression of p185 to the degree corresponding to the p185 reexpression level, but secretion of membrane-degrading gelatinases and invasion through the basement membrane preparation Matrigel by these cells were still inhibited by E1A. The data demonstrated that reexpression of p185 in neu+E1A cells can counteract the tumor-suppressing function of E1A but not completely recover the neu-induced metastatic phenotype. We conclude from these results that (a) repression of neu oncogene expression was indeed the molecular mechanism by which E1A suppressed tumor formation in neu-transformed 3T3 cells, and (b) suppression of metastasis by E1A in neu-transformed 3T3 cells was via multiple molecular mechanisms in addition to repressing neu. Our model system clearly demonstrated that tumorigenicity and metastasis are related but separable phenomena.

3T3 Cells↗
PubMed1993

Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A.

Amplification or overexpression of c-erbB-2/neu protooncogene, or both, occur frequently in many different types of human cancers and have been shown to correlate with decreased survival in ovarian cancer patients. We have previously found that the ovarian carcinoma cell line SK-OV-3 overexpresses c-erbB-2/neu mRNA. To further study the biological effect of c-erbB-2/neu overexpression in SK-OV-3 cells, we injected such cells i.p. into female nu/nu mice and found that this cell line forms extensive abdominal tumors and ascites. From the ascites in an injected mouse, we established the SKOV3.ip1 cell line and found that it expressed 2-fold more c-erbB-2/neu-encoded p185 proteins than the parental SK-OV-3 cells. When transformation phenotypes of SK-OV-3 and SKOV3.ip1 cells were compared, SKOV3.ip1 cells showed higher cell growth and DNA synthesis rates, formed more colonies in soft agar, produced larger s.c. tumors, and resulted in shorter survival of nu/nu mice after i.p. injection. These data indicate that the level of c-erbB-2/neu overexpression may correlate with the degree of malignancy in these ovarian carcinoma cells. Since we had previously shown that the adenovirus 5 E1A gene product can suppress transformation and metastatic properties induced by mutation-activated rat neu oncogene in mouse embryo fibroblast cells, we further examined whether E1A can abrogate malignancy in c-erbB-2/neu-overexpressing human ovarian cancer cells. We introduced the E1A gene into c-erbB-2/neu-overexpressing SKOV3.ip1 cells and found that the E1A-expressing ovarian cancer cell lines had decreased c-erbB-2/neu-encoded p185 expression and reduced malignancy, including a decreased ability to induce tumors in nu/nu mice. Therefore, we concluded that E1A is a tumor suppressor gene for c-erbB-2/neu-overexpressing human ovarian cancer cells and may be useful in developing therapeutic reagents for these human cancers.

Adenovirus E1A Proteins↗
PubMed1992

Substrate preference of metalloproteinases secreted by ts-110 Moloney murine sarcoma virus-transformed normal rat kidney cells.

In this study, the two forms of affinity-purified transformation-associated proteins (TAPs) (68 and 64 kD) were shown to have different substrate preferences. For the 68-kD TAP, the order of substrate preference was collagen types I, III, and V; fibronectin; gelatin; and collagen IV. For the 64-kD TAP, the order of substrate preference was collagen I, III, and V and gelatin. The 64-kD TAP did not cleave collagen IV and fibronectin. We also found a 71-kD metalloproteinase in the concentrated purified TAPs that reacted only weakly with a TAP monoclonal antibody and showed this substrate preference: collagen I, III, and V; gelatin; and collagen IV. Whether this 71-kD TAP is a new member of the rat metalloproteinase family will be investigated.

Animals↗
PubMed1992

Molecular cloning and characterization of v-mos-activated transformation-associated proteins.

Using monoclonal antibodies we previously detected two forms of transformation-associated proteins, a 64-kDa protein and a 68-kDa protein, in temperature-sensitive 110-Moloney murine sarcoma virus-mutant-transformed rat kidney 6m2 cells. The identity and functions of the transformation-associated proteins were previously unknown. By molecular cloning techniques and immunoscreening, we have isolated two cDNA clones (34A and 79B3) that were found by Western blot analysis to code for a monoclonal anti-transformation-associated protein antibody-reactive polypeptide of approximately 58 kDa. Limited restriction enzyme mapping indicated 34A and 79B3 are two different cDNA clones. The nucleotide sequence of 34A cDNA was determined, and a search of GenBank revealed that it is identical to that of rat transin-2. The deduced amino acid sequence of 34A shares 71% sequence identity with rat transin and 41-76% identity with six human metalloproteinases. The limited restriction enzyme mapping and partial nucleotide sequencing data indicated that 79B3 may be the rat transin gene. When either 34A cDNA or 79B3 cDNA was used as a probe in Northern blot analysis, one mRNA band of approximately 1.9 kilobases was detected in 6m2 cells grown at the permissive temperature of 33 degrees C, at which the cells exhibited transformation properties, and a much lower level in 6m2 cells grown at the nonpermissive temperature of 39 degrees C, at which the cells reverted to normal phenotypes. These results suggest that at 39 degrees C, these two genes were not transcribed at the same level as at 33 degrees C. Zymogram and Western blot analysis of 6m2 cells further confirmed that the 64- and 68-kDa proteins have metalloproteinase activities and that the synthesis of metalloproteinases was also temperature-sensitive. Apparently, the two proteins we formerly designated transformation-associated proteins are members of the rat transin gene family. Therefore, within v-mos transformed 6m2 cells, the absence of transformation-associated protein (metalloproteinase) synthesis at the nonpermissive temperature was due to the absence of transcription of two rat transin genes.

Amino Acid Sequence↗
PubMed1992

Adrenergic receptor-mediated increase of intracellular Ca2+ concentration in isolated bovine corneal epithelial cells.

1. We determined if Ca2+ is a second messenger for adrenergic receptor-effector coupling in bovine corneal epithelial cells. 2. Methoxamine (10(-5) M) selectively increased intracellular Ca2+ concentration (Cai) by 65%. This increase was only partially suppressed through the removal of extracellular Ca2+ or pretreatment with 10(-6) M verapamil. 3. The beta-adrenergic-mediated increases in Cai were entirely dependent on extracellular Ca2+. These increases were directly elicited through stimulation of adenylate cyclase because 10(-6) M isoproterenol and the active analogues of forskolin (10(-5) M) all elevated Cai. 4. Therefore, increases in Cai serve a second messenger function for alpha-1 and beta-adrenergic receptor-effector coupling.

Adenylyl Cyclases↗
PubMed1991

Prolonged exposure to N-methyl-D-aspartate increases intracellular and secreted somatostatin in rat cortical cells.

Somatostatin (SRIF) release from fetal rat cortical cells was stimulated by exposure to 10(-5) M N-methyl-D-aspartate (NMDA) (250 +/- 20% of basal at 96 h). A similar but much less potent effect was seen with kainate (KA) but not with quisqualate (Q) which inhibited SRIF release (KA 150 +/- 13%, Q 65 +/- 18% of basal at 96 h). Similar data were obtained for intracellular levels of SRIF. Dose-dependent experiments showed that the EC50 for the stimulatory action of NMDA was 2-3 x 10(-6) M with a Bmax of around 10(-5) M. At 10(-4) M KA and Q but not NMDA reduced tissue content and release of SRIF (KA: 47 +/- 14, 67 +/- 17%; Q: 36 +/- 13, 42 +/- 6% of basal for content and release, respectively). These findings indicate that cortical SRIF content and release is enhanced by exposure to NMDA but not by KA or Q. We suggest that SRIF-containing neurones are sensitive to glutamate damage through the activation of non-NMDA rather than NMDA receptors.

Animals↗
PubMed1991

Temperature-sensitive synthesis of a metalloproteinase in ts110-MSV-M-transformed NRK cells.

Previously, we reported that transformation associated protein (TAP) was over-expressed in the 6m2 line, but not in their normal counterparts (1,2). 6m2 is a culture of NRK cells transformed by the ts-110 mutant of MSV-M. The synthesis of TAP and the expression of transformation properties in the 6m2 cells are all temperature-sensitive (2; 3; 4). TAP is secreted as two polypeptides of 64 kD and 68 kD (P64 and P68) (2). Experiments were carried out to determine whether any metalloproteinase (MP) activity was associated with TAP. Results of zymograms indicated that the two forms of purified TAP (P64 and P68) had MP activity, using gelatin or collagen type IV as substrates. Serum-free medium (SFM) of 6m2 cells incubated at 33 degrees C also showed two bands of MP activity, while the corresponding SFM from 6m2 cells at 39 degrees C lacked such MP activity, indicating that the synthesis of MP was temperature-sensitive. The association of MP activity with the P64 and P68 bands of TAP (purified or in SFM) was confirmed by simultaneous Western blot analysis, which showed the reactivity of the two MP bands with monoclonal or polyclonal antibodies to TAP. Accordingly, what we previously designated as TAP is apparently one form of MP, which are known to be involved in tumor cell metastasis.

Animals↗
PubMed1990

Immunochemical comparison of transformation-associated protein and secreted phosphoprotein.

Transformation-associated protein (TAP) has been detected in MSV-M-transformed rat cell lines as glycosylated, weakly phosphorylated protein of molecular weight (Mr) 66,000 and 68,000. In the ts-MSV-M-transformed rat kidney cell line (6m2), the synthesis of TAP and the v-mos gene product is temperature-sensitive and accompanies the expression of transformation phenotypes. Therefore, TAP potentially plays a role in cellular transformation. On the other hand, SPP represents a family of glycosylated phosphoprotein with apparent Mr ranging from 42,000 to 69,000. SPP has been detected in osteoblasts and in avian and murine retrovirus-transformed rat and mouse epithelial cells. Therefore, the potential relatedness of TAP and SPP was studied. Using the 6m2 cells, we found that SPP was strongly phosphorylated and was synthesized at both the permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. By contrast, TAP was weakly phosphorylated, and was synthesized, as we found previously, only at the permissive temperature of 33 degrees C. Furthermore, in 35S-methionine incorporation studies, TAP became heavily labelled whereas SPP was not (consistent with its amino acid composition having few methionine residues). Using 125I-TAP in both immunoprecipitation and radioimmunoassays, it was found that an antiserum raised against SPP did not cross-react with 125I-TAP. Additionally, SPP has now been found in many human and rodent cells, while TAP thus far has only been detected in MSV-transformed rat cells. These data suggest that structurally, TAP and SPP are not closely related phosphoproteins.

Animals↗
PubMed1989

Growth hormone responses to growth hormone releasing factor in primary degenerative dementia.

The growth hormone (GH), thyroid-stimulating hormone (TSH), and prolactin (PRL) responses to growth hormone releasing factor (GRF) were investigated in 18 patients suffering from primary degenerative dementia (PDD) and in 20 age- and sex-matched normal elderly controls. There was no significant difference in the growth hormone response to GRF stimulation between patients and controls, and in neither subject group was there a demonstrable TSH or prolactin response to GRF. These findings indicate that the pathophysiology underlying the blunted growth hormone response to pharmacological challenge in PDD must lie at a suprapituitary level.

Aged↗
PubMed1989

Effect of cardiac arrest time on cortical cerebral blood flow during subsequent standard external cardiopulmonary resuscitation in rabbits.

Standard external cardiopulmonary resuscitation (SECPR) produces high cerebral venous and intracranial pressure peaks, low cerebral perfusion pressure, and low cerebral blood flow (CBF). Cerebral viability seems to require 20% of normal CBF, which SECPR cannot reliably generate. We tested the hypothesis that SECPR can produce adequate CBF if started immediately, but not if started after a long period of cardiac arrest (no flow, stasis). Cardiac arrest times of 1, 3, 5, 7 and 9 min were studied in rabbits. We measured unifocal cortical CBF with H2 clearance curves after saturation with H2 10%, O2 50% and N2O 40% by intermittent positive-pressure ventilation (IPPV). Measurements were made during spontaneous circulation (control condition), and then after resaturation immediately before induction of asystole by KCl i.v., and H2 clearance starting at end of arrest time during SECPR-basic life support with IPPV 100% and manual chest compressions (120/min) during asystole. Control cortical CBF was 30-40 ml/100 g brain per min. During asystole and SECPR, CBF greater than 20% normal was achieved only after no-flow of 1 min. After longer arrest (no-flow) times, CBF was less than 20% normal. Values were near zero after 7 and 9 min of cardiac arrest. Decrease in mean arterial pressures (MAP) produced by SECPR during asystole paralleled CBF values. Thus, the longer the preceding period of stasis, the lower the MAP and CBF generated by SECPR without epinephrine. This effect may be the result of anoxia-induced vasoparalysis and stasis-induced increased blood viscosity.

Animals↗
PubMed1989

Cytolysis by tumor necrosis factor is preceded by a rapid and specific dissolution of microfilaments.

Tumor necrosis factor (TNF) is cytotoxic to certain transformed cells, whereas normal cells are resistant to its effects. The resistance of normal cells can often be overcome by treatment with inhibitors of transcription or translation such as actinomycin D or cycloheximide (CHI), suggesting that normal cells produce a protein(s) that protects them from TNF-induced cytolysis. In this report, we examine the mechanism of cytolysis in a 3T3-like mouse cell line, C3HA, which was sensitized to TNF by treatment with CHI. We found that an early change in TNF/CHI-treated cells was a significant loss of stress fibers in perinuclear areas of the cytoplasm. The disruption of microfilaments, which was observed within 15 min of treatment, was not seen in untreated cells or in cells treated with either TNF or CHI alone. The dissolution of microfilaments spread peripherally over time and preceded other TNF/CHI-induced effects such as cytoplasmic "boiling," decrease in cell volume, and lysis of the plasma membrane. The breakdown of stress fibers occurred without a change in microtubules or intermediate filaments. Cytochalasin E, which disrupts microfilaments, induced cytolysis of TNF-treated cells even in the absence of CHI; however, demecolcine, which depolymerizes microtubules, did not sensitize cells to TNF. We propose that the TNF-induced cytolysis of certain cell types is preceded by a selective disruption of the microfilament lattice.

Actin Cytoskeleton↗
PubMed1988

Calcium retention and increased vascular reactivity caused by a hypothalamic sodium transport inhibitor.

1. Using a previously established method of isolating an active-sodium-transport inhibitor (ASTI) from hypothalamic cell culture medium, the inhibitor was isolated and partially purified from sequential passages through Sephadex G-25 and h.p.l.c., and its effects on de-endothelialized rabbit aortic strips were investigated. 2. ASTI caused a cumulative concentration-dependent increase in tension which reversed slowly after wash, and the wash showed an identical effect on fresh strips. 3. Ouabain, used as a control, also caused a concentration-dependent increase in tension which reached a plateau at a concentration of 10 mmol/l. Both ouabain and ASTI caused a significant potentiation of the vasoconstrictor effect of noradrenaline at concentrations of 1 nmol/l-0.1 mmol/l. 4. Both ASTI and ouabain caused a significantly greater (P less than 0.01) calcium retention than control medium in aortic strips. 5. Incubation of ASTI with prolidase, chymotrypsin and carboxypeptidase A destroyed the vasoconstrictor effects as well as its inhibitory effects on sodium, potassium-dependent adenosine triphosphatase and sodium efflux from erythrocytes, but leucine aminopeptidase was ineffective. 6. These studies suggest that hypothalamic cells in culture release a peptidic inhibitor of active sodium transport which increases vascular reactivity, potentiates vasoconstrictor effects of noradrenaline and causes calcium retention.

Animals↗
PubMed1987

A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement.

The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.

Benzofurans↗
PubMed1987

Problems and pitfalls in the isolation of an endogenous Na+, K+-ATPase inhibitor.

Plasma from volume-expanded and salt-loaded hypertensive animals and from patients with essential hypertension has been reported to inhibit Na+, K+-adenosine triphosphatase (ATPase). Inhibition of the sodium pump in vascular smooth muscle caused by such a circulating factor could increase vascular tone and sensitivity to vasoactive agents, and thereby result in arterial hypertension. Numerous efforts in the past failed to isolate the putative factor from urine and plasma. Recent studies have suggested that the hypothalamus is an important source of an endogenous Na+, K+-ATPase inhibitor, but its isolation from the tissue extracts has been rendered difficult by the presence of other cellular constituents that cause artifactual interference with the assays and purification procedures. Using an alternative approach of isolating the inhibitor from culture medium, we found that dispersed fetal rat hypothalamic neurons in a capillary culture system release a heat-stable, peptidic, low-molecular-weight, active sodium transport inhibitor that causes a reversible increase in vascular tone, sensitizes vascular smooth muscle to the vasoactive effect of norepinephrine, and possesses several characteristics of the putative endogenous digitalislike factor. This inhibitor may be a chemical mediator linking kidney, brain, and cardiovascular system in the genesis of experimental volume-expanded and salt-loaded hypertension and human essential hypertension.

Animals↗