Drinking green tea is associated with decreased frequency of cancer development. This review outlines the wide range of mechanisms by which epigallocatechin gallate (ECGC) and other green and black tea polyphenols inhibit cancer cell survival. EGCG suppressed androgen receptor expression and signalling via several growth factor receptors. Cell cycle arrest or apoptosis involved caspase activation and altered Bcl-2 family member expression. EGCG inhibited telomerase activity and led to telomere fragmentation. While at high concentrations polyphenols had pro-oxidative activities, at much lower levels, anti-oxidative effects occurred. Nitric oxide production was reduced by EGCG and black tea theaflavins by suppressing inducible nitric oxide synthase via blocking nuclear translocation of the transcription factor nuclear factor-kappaB as a result of decreased IkappaB kinase activity. Polyphenols up- or down-regulated activity of a number of key enzymes, including mitogen-activated protein kinases and protein kinase C, and increased or decreased protein/mRNA levels, including that of cyclins, oncogenes, and tumor suppressor genes. Metastasis was inhibited via effects on urokinase and matrix metalloproteinases. Polyphenols reduced angiogenesis, in part by decreasing vascular endothelial growth factor production and receptor phosphorylation. Recent work demonstrated that EGCG reduced dihydrofolate reductase activity, which would affect nucleic acid and protein synthesis. It also acted as an aryl hydrocarbon receptor an-tagonist by directly binding the receptor's molecular chaperone, heat shock protein 90. In conclusion, green and black tea polyphenols act at numerous points regulating cancer cell growth, survival, and metastasis, including effects at the DNA, RNA, and protein levels.

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.

Canine tracheal mucin glycoprotein was isolated from beagle dogs fitted with tracheal pouches. Following exclusion chromatography on Sepharose CL-4B, noncovalently associated proteins were further resolved by dissociative density gradient centrifugation in CsBr-guanidinium chloride, and the mucin was then extracted with chloroform-methanol. The delipidated high-density product obtained had a nominal molecular weight of about 10(6) and an overall composition characteristic for a mucin glycoprotein, viz., a high content of serine and threonine, about 80% carbohydrate by weight, the absence of mannose or uronic acid, measurable ester sulfate, and a Pronase-resistant domain of molecular weight (1.75-3.0) X 10(5) which contains essentially all of the saccharide residues. Noncovalently bound lipid amounted to 6-10% by weight and was primarily cholesterol and cholesteryl esters. Cleavage of disulfide bonds by performic acid oxidation resulted in the release of a protein (Mr 65,000) not otherwise resolved by sodium dodecyl sulfate gel electrophoresis or the purification scheme.

The effects of concanavalin A and ricin (RCAII, Mr 65,000) on [3Hlthymidine incorporation into human neuroblastoma IMR-32 DNA showed reduction of total DNA synthesis to 50% and 70% of control, respectively. Two DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) activities (a and P) involved in the biosynthesis in vitro of DNA were separated by sucrose density gradient centrifugation from IMR-32 cell homogenate. The DNA polymerase a activity was also purified by selective precipitation with polyethylene glycol (Mr 6000) followed by agarose-concanavalin A column chromatography. The activities of both DNA polymerases were examined at various concentrations of mutagenic and nonmutagenic plant agglutinins and the toxin ricin. Concanavalin A and ricin specifically inhibited DNA polymerase a activity (activity reduced to 19% and 10%, respectively), whereas DNA polymerase P activity was inhibited (reduced to 16%) by red kidney bean agglutinin (PHA-P). Various plant lectinst are toxic to human and other animal cells grown in vivo (2, 3) or in vitro (3-5). The tumor-suppressive effects of two lectins, concanavalin A (Con A) (3-5) and phytohemagglutinin (PHA) (6-8), and differential toxic effects of a few plant toxins such as ricin (RCAII, a glycoprotein; Mr 65,000) and abrin (a toxin isolated from Abrus precatorius; Mr 63,000) (9-11) have been demonstrated on tumor and transformed cells. It has been proposed that ricin interferes in the peptide chain elongation step by interacting with 60S ribosomal subunits (12); furthermore, the inhibitory property increases after treatment of the toxin with 2-mercaptoethanol (13). The cell surface-binding properties, but not the protein synthesisinhibitory properties, of ricin (14) New England Nuclear (1 Ci = 3.7 X 1010 becquerels). RCAI, RCA11, peanut agglutinin (PNA), and soybean agglutinin (SBA) were purchased from E. Y. Laboratories (San Mateo, CA). Wheat germ agglutinin (WGA) and Con A were purchased from Vector Laboratories and Sigma, respectively. PHA-P and PHA-M were purchased from Difco. Abrus precatorius agglutinin (APA) was a gift sample from A. Sen of The Bose Institute (Calcutta, India).Isolation of DNA Polymerases a and ft. Human neuroblastoma clone IMR-32 cells were purchased from the American Type Culture Collection and maintained in our laboratory as described (26)(27)(28). Confluent monolayers (5 to 7 X 106 cells per 250-ml Falcon plastic flask) were harvested with phosphate-buffered saline [7.0 mM potassium phosphate/0.14 M NaCl buffer, pH 7.2 (P1/NaCl)l containing 0.1% EDTA for enzymatic studies. A 25% (vol/vol) homogenate of cells in 0.32 M sucrose containing 10 mM Tris1HCI buffer (pH 7.8) was obtained, and DNA polymerase a and 3 activities were subsequently separated on a 5-20% continuous sucrose gradient containing 10 mM Tris-HCl (pH 8.0), 1 mM 2-mercaptoethanol, and 100 mM KCl as described (27). The gradient (5.0 ml) was centrifuged for 16 hr in a Beckman SW 50.1 swingingbucket rotor at 149,000 X g. Fractions (0.25 ml) were collected from the bot...

Three forms of DNA polymerase (pol) a from human neuroblastoma IMR-32 were separated by DEAE column chromatography. All sedimented at -7 S in 5-20% continuous sucrose density gradients. All were heat labile, with pol a2 the most (90% inactivated) and pol a3 the least (50% inactivated) sensitive to heating for 5 min at 50TC. pol a, and a2 efficiently utilized activated calf thymus DNA as template. The most active form, pol a2, used both poly(dA)-(dT)j2_j8 and poly(dT)-(dA)12_18 as template at equal rates. Differential inhibition of DNA polymerase at activities was examined in the presence of ricin, hemin, and a nonhistone chromatin protein. All three polymerases were inhibited by both ricin (nonreduced) and hemin,. with pol a2 the most (80-90%) and pol a3 the least (60%) sensitive in each case. In contrast, only pol a2 and a3 activities were inhibited (80-85%) by rat liver nonhistone chromatin protein.The occurrence of multiple forms of DNA polymerase (pol) a (DNA nucleotidyltransferase, EC 2.7.7.7) activity in calf thymus and rat liver (1-6), mouse myeloma (7-9), mouse mastocytoma (10), and rat ascites hepatoma cells (11) has been reported in recent years. Very little work has been done on characterization of the various forms of DNA pol a from cultured human cells until recently (12)(13)(14). Moreover, it is not known whether the different forms of DNA pol a have different reaction rates during replication of eukaryotic genomes. Each of these DNA pol a forms can generate low molecular weight species (6). Whether the occurrence of multiple forms is due to some modification of a common catalytic subunit or to the presence of different primary structures has yet to be established with purified enzymes isolated from normal and tumor cells. Tryptic peptide maps of individual subunits of two forms of highly purified mouse myeloma DNA pol a (9) indicate no structural homology between the two forms. One ofthese forms contains both 3'--5' and 5'--3' exonuclease activities (9). The different template specificities of different forms of DNA pol a in mouse myeloma solid tumors (9, 15) suggest that these forms may play different biological roles in the recognition of base sequences, thereby regulating cell division and differentiation.We have reported the inhibition of RNA-primed (20). A mixture of template and primer (20:1) in 50 mM NaCl/ 5 mM MnCl2 was heated for 10 min at 550C, maintained at room temperature for 1 hr, and stored at -18'C. Subscripts denote chain lengths of polynucleotide primers.Isolation of Multiple Forms of DNA pol a. Human neuroblastoma clone IMR-32 cells were maintained in our laboratory as described (17,21). Confluent monolayers (5-7 x 106 cells per 250-ml Falcon plastic flask) were harvested with 7.0 mM potassium phosphate, pH 7.2/0.14 M NaCV0. 1% EDTA. To isolate and purify DNA pol -x from neuroblastoma, IMR-32 frozen cells were thawed and homogenized in 50 mM Tris'HCl, pH 7.5/1 mM MgClJ6 mM KCV1 mM 2-mercaptoethanol. The homogenate was sonicated for four 15-sec intervals at 40C. The son...