Mapping the binding site of colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354.

2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with beta-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of beta-tubulin in protofilaments (Nogales, E. , Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203), we found two potential binding sites. At one, entirely encompassed within beta-tubulin, the C2- and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the alpha/beta interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.

6beta-Acyloxy(nor)tropanes: affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors.

A series of esters of 6beta-hydroxynortropane and the N-methyl analogue 6beta-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m(1)-, m(2)-, m(3)-, and m(4)-muscarinic receptors in transfected cell membranes and on native M(1)-muscarinic receptors in rat brain membranes and native M(2)-muscarinic receptors in rat heart membranes. Most 6beta-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m(1)- and native M(1)-receptors, indicative of agonist activity. 6beta-Acetoxynortropane had K(i) values versus oxotremorine-M binding at m(1)-, m(2)-, and m(4)-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6beta-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6beta-Acyloxy(nor)tropanes and classical muscarinic agonists, such as muscarine and oxotremorine, had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at native M(2)-muscarinic receptors of heart, but not at transfected m(2)-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m(3)-receptors, since significant oxotremorine-M binding could not be detected. 6beta-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m(4)-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.

Allosteric modulators of the AMPA receptor: novel 6-substituted dihydrophthalazines.

Novel analogs of the allosteric AMPA receptor modulator SYM 2206 have been prepared. Structure/activity correlations of these novel analogs and other dihydrophthalazines (DHPs) reveal the important contribution of the heteroatom-based aryl substituents in this class of noncompetitive inhibitors. One of the analogs (6, SYM 2189) is equipotent with the early series, but with reduced sedation.

Long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.

The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.

HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium.

The E6 and E7 genes of HPV-16 or HPV-18 both are necessary for effective immortalization of primary human genital keratinocytes. To analyse the individual role of E6 and E7 genes in dysregulating cell growth, we cloned the HPV-16 E6, E7 and E6/E7 genes into retroviruses. Primary human keratinocytes (PHK) were then infected with these retroviruses and selected in differentiation-inducing medium (high calcium and serum). The E6/E7 retroviruses were the most effective at inducing differentiation-resistant colonies. Intermediate numbers of colonies were induced by E6 and low numbers by E7. Interestingly, only cultures infected with E7 and E6/E7 retroviruses showed a significant proportion of cells progressing into the S phase, consistent with our earlier studies showing that E7 is required for the efficient immortalization of genital keratinocytes. Accompanying this entry into S phase, the E7 or E6/E7 transduced cells expressed high levels of cyclins A, B and E, but lower levels of cyclin D. In addition, cdc-2, cdk-2 and cdk-4 were also increased. No significant differences were detected in the expression of c-myc and c-fos between the vector and any of the transduced cells. Keratinocytes infected with the E7 retrovirus exhibited decreased levels of Rb protein and increased levels of p53, whereas cells infected with E6-expressing retroviruses displayed normal levels of Rb protein and decreased levels of p53. Finally, E7 induced a three-fold increase in bcl-2 expression. Our results indicate that the HPV-16 E7 gene alone is sufficient to bypass keratinoctye growth arrest induced by serum and calcium exposure and that the discordant expression of several cell regulatory proteins accompanies this unregulated proliferation.

6beta-Acetoxynortropane: a potent muscarinic agonist with apparent selectivity toward M2-receptors.

A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a Chinese herb, were synthesized. 6beta-Acetoxynortropane (5) had weak affinity (Ki 22 microM) for central (M1) muscarinic receptors in a [3H]quinuclidinyl benzilate binding assay but had extremely high affinity (Ki 2.6 nM) and selectivity for M2-muscarinic receptors expressed in CHO cells. It had 13-fold lower affinity for M4-receptors, 260-fold lower affinity for M3-receptors, and 8200-fold lower affinity for M1-receptors expressed in CHO cells. The 6beta-carbomethoxy analogue (14) of baogongteng A had only weak affinity for M2-muscarinic receptors, as did 6beta-carbomethoxynortropane (13) and 6beta-acetoxytropane (4). In transfected CHO cells, 6beta-acetoxynortropane (5) was an agonist at M2-receptors, based on a GTP-elicited decrease in affinity, and a full agonist with an IC50 of 11 nM at M4-receptors, based on inhibition of cyclic AMP accumulation, while being a full agonist at M1-receptors with an EC50 of 23 nM and a partial agonist at M3-receptors with an EC50 of 3.6 nM, based in both cases on stimulation of phosphoinositide breakdown. All of the 16 tropane derivatives had weak affinities for central alpha4beta2-nicotinic receptors with 6beta-carbomethoxynortropane (13) having the highest affinity, which was still 150-fold less than that of nicotine. 6beta-Acetoxynortropane (5) represents a potent muscarinic agonist with apparent selectivity toward M2-receptors.

Induction of apoptosis in human keratinocytes containing mutated p53 alleles and its inhibition by both the E6 and E7 oncoproteins.

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. We have investigated the response of immortalized human keratinocytes (HaCaT) bearing mutated alleles of p53 to genotoxic stress and the effect of human papillomavirus (HPV) 16 E6 and E7 on this response. UVC irradiation triggered HaCaT's cell death with several characteristics of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and the appearance of cells with a low content of DNA (categorized as sub-G1 by cell sorter analysis). This response was accompanied by accumulation of cells in S phase of the cell cycle. HaCaT cells infected with retroviruses carrying HPV16 E6 or E7 showed a significant reduction in their apoptotic response, which was not observed in cells infected with the LXSN vector DNA-carrying virus. Reduced apoptosis in HaCaT cells expressing E6 or E7 also was observed after treatment with the alkylating agent mitomycin C. Western blot analysis of p53 and p21/WAF-1/CIP-1, a downstream effector of p53, did not reveal any changes in the levels of these proteins after UVC irradiation in either HaCaT cells or HaCaT cells expressing HPV16 E6 or E7.

Total syntheses and anticholinesterase activities of (3aS)-N(8)-norphysostigmine, (3aS)-N(8)-norphenserine, their antipodal isomers, and other N(8)-substituted analogues.

N(8)-Benzylesermethole (6) was prepared from 5-methoxytryptamine (1) in five steps. Resolution of compound 6 by dibenzoyl- and ditoluyltartaric acid provided enantiomers (-)- and (+)-7. After demethylation, reaction with isocyanates and catalytic debenzylation over hydrogen, the total syntheses of (-)- and (+)-N(8)-norphysostigmine [(-)- and (+)-11] and (-)- and (+)-N(8)-norphenserine [(-)- and (+)-12] were accomplished, (-)-N(8)-Norphysostigmine [(-)-11] and (-)-N(8)-norphenserine [(-)-12] were also obtained by transformations of natural physostigmine [(-)-13] and phenserine [(-)-14] prepared from (-)-13. The absolute configurations and optical purity of compounds (-)-11, (-)-12, (+)-11, and (+)-12 were confirmed by a comparison of their optical rotations with those of the compounds synthesized from physostigmine [(-)-13]. The anticholinesterase activities of N(8)-nor- and N(8)-substituted analogues, (-)- and (+)-9, -10, -11, -12, 15, and 16, were compared with those of physostigmine [(-)- and (+)-13] and phenserine [(-)- and (+)-14] and are reported.

Pharmacological modulation of Alzheimer's beta-amyloid precursor protein levels in the CSF of rats with forebrain cholinergic system lesions.

Abnormal deposition and accumulation of Alzheimer's amyloid beta-protein (A beta) and degeneration of forebrain cholinergic neurons are among the principal features of Alzheimer's disease. Studies in rat model systems have shown that forebrain cholinergic deficits are accompanied by induction of cortical beta-amyloid precursor protein (beta-APP) mRNAs and increased levels of secreted beta-APP in the CSF. The studies reported here determined whether the CSF levels of secreted beta-APP could be altered pharmacologically. In different experiments, rats with lesions of the forebrain cholinergic system received injections of vehicle, a muscarinic receptor antagonist scopolamine, or one of two cholinesterase inhibitors - diisopropyl phosphorofluoridate (DFP) or phenserine. Scopolamine was administered to determine whether the levels of beta-APP in the CSF could be increased by anticholinergic agents. The cholinesterase inhibitors were administered to determine whether the forebrain cholinergic system lesion-induced increases in CSF beta-APP could be reduced by cholinergic augmentation. Scopolamine administration led to a significant increase in the CSF levels of secreted beta-APP in sham-lesioned rats. Phenserine, a novel, reversible acetyl-selective cholinesterase inhibitor, significantly decreased the levels of secreted beta-APP in the CSF of forebrain cholinergic system-lesioned rats whereas DFP, a relatively non-specific cholinesterase inhibitor, failed to affect CSF levels of secreted beta-APP. These results suggest that the levels of secreted beta-APP in the CSF can be pharmacologically modulated but that this modulation is dependent upon the status of the forebrain cholinergic system and the pharmacological properties of the drugs used to influence it.

The human papillomavirus E6/E7 genes induce discordant changes in the expression of cell growth regulatory proteins.

The E6/E7 oncoproteins of human papillomavirus (HPV) types 16 and 18 are responsible for the efficient immortalization of human genital keratinocytes and we have recently reported that such immortalized cells display alterations in the expression of cyclin A, cyclin B, and cdc-2. To determine whether these alterations were the consequence of E6/E7 protein expression or whether they resulted from the process of cellular immortalization, we multiply-infected primary genital keratinocytes with a retrovirus expressing the HPV-18 E6/E7 genes and examined the cells for acute, pre-immortalization changes in several critical cell growth regulatory proteins including cyclin A, cyclin B, cdc-2, p53 and c-myc. In addition, we simultaneously evaluated the expression of the E6/E7, bcl-2 and involucrin genes to determine whether there were accompanying alterations in the expression of viral genes or in cellular genes related to cell apoptosis and the state of keratinocyte differentiation. The cell cycle regulating proteins (cyclin A, cyclin B, cdc-2 and p53) change significantly within days after retroviral infection. Cyclin B and cdc-2 increase over 4-fold by three passages and remain relatively constant thereafter through passage 21, whereas the levels of p53 protein decrease 25% by passage three. Increases in the expression of cyclin A, cyclin B and cdc-2, and decreases in p53 are therefore among the earliest observable changes in cell regulatory proteins following E6/E7 gene expression and may be important contributors to the development of cell immortalization. The expressions of viral E6/E7 genes, c-myc, bcl-2 and involucrin exhibit progressive changes with increased passage numbers until passage 21, presumably reflecting the selective outgrowth of immortalized cells.

Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine.

The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 A. using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[14C] Carbonyl)-3- demethylthiocolchicine ([14C]3CTC). This beta-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)- 3-demethythiocolchicine ([14C]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radio-labeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [14C]3CTC and beta-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 A of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 A distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.

Maze learning in aged rats is enhanced by phenserine, a novel anticholinesterase.

A new generation of cholinesterase inhibitors is expected to overcome some limitations of the therapeutic use of anticholinesterases. Phenserine is a long-acting and selective inhibitor of acetylcholinesterase with a preferential brain uptake. We have assessed the effects of chronic phenserine tartrate treatment on performance of aged Fischer-344 rats in the 14-unit T-maze. Phenserine (1-3 mg kg-1, i.p.) treatment for 5 days significantly reduced the number of errors made in the Stone maze. Other performance variables were also improved. No side effects were noted across 5 days treatment at doses of 1-2 mg kg-1. Phenserine can therefore improve the performance of aged rats in this complex maze task without producing obvious side effects.

Integrin expression and function in HPV 16-immortalised human keratinocytes in the presence or absence of v-Ha-ras. Comparison with cervical intraepithelial neoplasia.

Keratinocytes express several receptors of the integrin family which regulate both adhesion and differentiation. We have investigated whether HPV immortalisation, which changes the growth and differentiation properties of keratinocytes, is associated with altered integrin expression or function. We compared two HPV 16-immortalised lines of human keratinocytes, up and vp, with the normal keratinocyte strains, u and v, from which they were derived and with upr, obtained by transfection of up with viral Harvey ras. Immunofluorescence, immunoprecipitation and flow cytometry demonstrated that up and vp had lower levels of integrins than u and v, the reduction in up being greater than in vp. Up and vp also had reduced levels of mRNA encoding the beta 1 integrin subunit. Reduced expression of the alpha 5 beta 1 and alpha 2 beta 1 integrins was correlated with reduced adhesion to fibronectin and collagen in up but not in vp and there were no significant differences between the normal and immortalised cells in adhesion to laminin. Reduced integrin expression was correlated with decreased motility, up showing a greater reduction in motility than vp. Introduction of activated ras into up had no effect on integrin levels, cell motility or tumorigenicity in nude mice; the only difference between up and upr was that upr showed increased adhesion to fibronectin. Examination of eight biopsies of cervical intraepithelial neoplasias with evidence of HPV infection revealed reduced or discontinuous integrin expression in the most severe lesions. We conclude that both in vivo and in culture keratinocytes the impaired differentiation that is associated with the presence of HPV is correlated with reduced integrin expression.

Elevated expression and activity of mitotic regulatory proteins in human papillomavirus-immortalized keratinocytes.

The E6 and E7 proteins of human papillomavirus (HPV) types 16 and 18 are expressed in cell lines derived from cervical cancers and can immortalize primary human keratinocytes. Since expression of E6/E7 has been shown to induce mitotic defects and karyotype instability in primary human cells, we investigated the effect of these viral oncoproteins on the expression and activity of mitotic regulatory proteins. Primary human keratinocytes immortalized by the entire genome or by only the E6/E7 genes of HPV types 16 and 18 displayed 5- to 20-fold increases in the abundance of p34cdc2, cyclin B and cyclin A when compared with normal parental cells. Results obtained from normal and immortalized cells that were derived from identical single donors were similar to those from mixed donor cultures. Increased protein levels were achieved without corresponding increases in mRNA, indicating alterations in translational and/or post-translational control. The histone H1 kinase activities associated with these regulatory proteins were also elevated, but to a lesser extent than the protein levels. Because p34cdc2, cyclin B and cyclin A regulate the entry into and exit from mitosis, increased expression and activity of these proteins could contribute to the mitotic defects and chromosomal aberrations associated with HPV-induced immortalization.

Keratinocytes immortalized by human papillomavirus-18 exhibit alterations dependent upon host genetic background and complexity of viral genes transfected.

Keratinocytes immortalized by the human papillomaviruses (HPVs) vary in cell morphology, growth properties, resistance to inducers of terminal differentiation, and karyotype. To determine the contribution of the host cell genetic background and the HPV genes to these cellular alterations, we have generated and characterized 6 human keratinocyte lines from two different newborn foreskins (A and B) using either the full-length HPV18 genome or the isolated HPV18 E6/E7 genes. The growth properties of the immortalized lines were found to correlate with the complexity of HPV genes present in the transfected vector. Interestingly, cell lines established from foreskin A revealed common chromosomal alterations regardless of the HPV construct utilized for immortalization, and these karyotypic changes differed from those observed in cell lines established from foreskin B, which exhibited their own characteristic aneuploid profile. Thus, chromosomal alterations of HPV-immortalized cells are in part determined by the host genetic background.

Clonal growth and origin of two human keratinocyte cell lines transformed by human papillomavirus type 16 DNA.

Two human keratinocyte cell lines transformed by human papillomavirus type 16, designated Vp and Up, were compared for their clonal growth potential and clonal origin. Up showed greater anchorage-independent growth in soft agar and higher efficiency of single-cell colony formation than Vp (24.3% compared to approximately 10%). The clonal growth potential of these two cell lines was not related to the level of HPV16 gene expression. Fourteen single cell clones of the Vp and 24 of the Up were selected, propagated and analyzed by Southern and Northern blot analysis. Clonal variations existed among subclones of each cell line and between the two cell lines. These variations included cell morphology, growth potential, and expression levels of involucrin (a differentiation marker of keratinocytes) and of HPV16 mRNAs. The Vp and Up cell lines also showed different patterns of HPV16-DNA integration and RNA transcription. However, all subclones of Vp and subclones of Up displayed identical HPV16 DNA integration and RNA expression patterns. The results suggest that both cell lines were monoclonal in origin and that the host genetic factors play an essential role in determining cell clonality.

Cotransfection of HPV-18 and v-fos DNA induces tumorigenicity of primary human keratinocytes.

Human keratinocytes were cotransfected with the FBJ/R v-fos oncogene and the HPV-18 genome and selected on the basis of their resistance to inducers of terminal differentiation. Unlike keratinocytes immortalized by HPV-18 DNA alone, the HPV-18/v-fos transformants exhibited prominent intracellular vacuolization and formed progressively growing, squamous cell tumors when injected subcutaneously into nude mice. Cytogenetic analysis of two clonally selected transformed cell lines (18/fos clone 1 and 18/fos clone 2) revealed minimal alterations in karyotype, although a consistent rearrangement of chromosome 10, iso(10q), was observed. Further analysis of 18/fos clone 1 confirmed the expression of the fos gene as well as the HPV-18 E7 protein. Alterations in fos gene expression, which appear to be an important facet of epidermal differentiation in vivo, could potentially contribute to the malignant progression of HPV immortalized cells.

Changes in type I keratin expression associated with HPV16 transformation of human epidermal keratinocytes.

We have used immunoblotting to compare expression of type I keratins in two strains of normal human epidermal keratinocytes (v and u) and their HPV16-transformed derivatives (vp and up). The levels of keratins 14 and 17 were similar in all four cell strains, whereas keratins 18 and 19 were more abundant in vp and up than in the normal parental keratinocytes. Keratin 13 was more abundant in the transformed cells than in the parentals; in addition, expression in v was higher than in u, and expression in vp was higher than in up, suggesting strain-specific variation in expression. Keratin 16 was the only keratin to be more highly expressed in v and u than in vp and up; this is consistent with the reduced capacity of the transformants for stratification and terminal differentiation. Double-label immunofluorescence of vp and up showed that more cells expressed involucrin than keratin 16. We conclude that HPV16 transformation results in marked changes in keratin expression. The increased expression of keratin 18, a keratin that is normally expressed in simple epithelia, fits well with reports of increased keratin 18 expression in invasive squamous cell carcinomas of skin and other keratinocyte-derived tumours.

Differential effect of tumor necrosis factor on proliferation of primary human keratinocytes and cell lines containing human papillomavirus types 16 and 18.

Keratinocytes immortalized by human papillomaviruses (HPV) 16 and 18 are partially resistant to the inhibition of proliferation exerted by transforming growth factor-beta (TGF-beta). To determine if this finding reflects a generalized resistance to inhibitory cytokines, we studied the effect of tumor necrosis factor-alpha (TNF-alpha) on subconfluent cultures of both normal and HPV-immortalized human foreskin keratinocytes. Whereas primary and HPV-16-immortalized keratinocytes were sensitive to TNF-alpha, HPV-18-immortalized keratinocytes (and those immortalized by simian virus 40) were resistant to the inhibitory effects of this cytokine. The ability of HPV-18 to induce a more resistant phenotype correlated with its more potent in vitro transforming activity and its apparent association with more aggressive tumors. Interestingly, the state of TNF-induced growth inhibition in normal or HPV-16-immortalized keratinocytes was not accompanied by a reduction in the expression of c-myc RNA or protein. This contrasts sharply with the ability of TGF-beta to inhibit c-myc RNA expression in normal cells. Evidently, the resistance of HPV-immortalized keratinocytes to TNF-alpha and TGF-beta proceeds along different regulatory pathways.