We report isolation of a novel tumor-reversing gene, tentatively named SVS-1, encoding a protein of 820 amino acids with localization on the plasma membrane as a type I transmembrane protein.

(1-4) Class I enzymes (topo I, EC 5.99.1.2) act by making a transient break in one DNA strand, allowing the DNA to swivel and release torsional strain, changing the linking number by steps of one.(2,4) Class II enzymes (topo II, EC 5.99.1.3) make transient breaks in both strands of one DNA molecule, allowing the passage of another DNA duplex through the gap, changing the linking number by steps of two.(1-3) These enzymes are crucial for cellular genetic processes such as DNA replication, transcription, recombination, and chromosome segregation at mitosis.It has long been accepted that topos are valuable targets of cancer chemotherapeutics.(2-5) Several classes of topo inhibitors have been introduced into cancer clinics as potent anticancer drugs, including camptothecin (CPT) derivatives (e.g. irinotecan and topotecan) inhibiting topo I (4) and anthracyclines (e.g. doxorubicin and mitoxantorone), epipodophyllotoxins (e.g. etoposide , aminoacridines (e.g. m-AMSA) and ellipticines targeting topo II.(4,5) These agents are active in both hematological and solid malignancies. The activity of these agents is thought to result from stabilization of the DNA /topo cleavable complex (CC), an intermediate in the catalytic cycle of the enzymes, (2,5,6) resulting ultimately in apoptosis. A number of new topo inhibitors have recently been reported that do not stabilize CC. Thus, two general mechanistic classes of topo inhibitors, especially for topo II, have recently been described: (7) (1) classical topo 'poisons' that stabilize CC and stimulate single-or double-strand cleavage of DNA, such as CPT and its derivatives, indolocarbazoles for topo I, and for topos I and II; and (2) catalytic inhibitors that prevent the catalytic cycle of the enzymes at steps other than cleavage intermediates, such as dioxopiperazines, (7,9,10) aclarubicin, (11) intoplicin, (12) and F11782.(13) Some of these compounds are dual inhibitors of topos I and II. The catalytic inhibitors of topo II, merbarone (14,15) and dioxopiperazines, (9,10) have been extensively studied and have been shown to inhibit the reopening of the closed clamp formed by the enzyme around DNA by inhibiting the ATPase activity of the enzyme, thus sequestering the enzyme within the cell. (7,16,17)

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It has been reported that low-power laser irradiation (LLI) can modulate various biological processes including cell proliferation. Some reports suggest that LLI interferes with the cell cycle and inhibits cell proliferation, while others suggest that LLI has a stimulatory effect. Mechanisms underlying the effects of LLI remain unclear. Since the effects of LLI on cancer cells are not well understood, with the aim of developing an LLI therapy for malignant glioblastoma, we investigated the effects of LLI on the cell proliferation of the human-derived glioblastoma cell line A-172. Glioblastoma cell cultures were irradiated with a diode laser at a wavelength of 808 nm and the effects on cell viability and proliferation were examined. Cell counting at 24 and 48 h after irradiation showed that LLI (at 18, 36 and 54 J/cm(2)) suppressed proliferation of A-172 cells in a fluence-dependent manner (irradiation for 20, 40 and 60 min). A reduction in the number of viable cells was also demonstrated by a fluorescent marker for viable cells, calcein acetoxymethylester (calcein-AM). The reduction in cell viability was not associated with morphological changes in the cells or with necrotic cell death as demonstrated by propidium iodide staining. LLI also had little effect on cell proliferation as shown by 5-bromo-2'-deoxyuridine staining. We discuss possible mechanisms underlying the suppressive effect of 808-nm LLI on the viability of human-derived glioblastoma A-172 cells.

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.

The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virusinfected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO2 and of decarboxylated S-adenosylmethionine (dSAM). The K, for SAM is 38 micromolar. The reaction is not stimulated by Mg' or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5. of Lathynrs sativus (19), a small amount of activity was concentrated and purified by affinity chromatography. The stoichiometry of the reaction was not determined nor was the amount of activity related to the amount of spermidine synthesized in the plant. This activity, as well as that found in mung bean sprouts (6), was shown to be Mge' dependent, as are some bacterial SAM decarboxylases. The levels ofbiosynthetic activity detected in extracts of these plant materials were extremely low and may have arisen from bacterial contamination. On the other hand, the activity of SAM decarboxylase isolated from corn seedlings (20) has been reported to be independent of Mge' or putrescine, as is the enzyme we have now isolated from Chinese cabbage. Nevertheless, the low level of activity of the corn enzyme, by comparison to the amount of spermidine found in plants, has similarly raised the problem of the significance of this activity.One product of the SAM decarboxylase reaction, dSAM, is difficultly available. Despite its recent synthesis, an extinction coefficient has not been published (15) and a direct measurement of the stoichiometry of the decrease of SAM and appearance of dSAM has not previously been attempted. For this reason the decarboxylation reaction catalyzed by several presumed SAM decarboxylases cannot be considered unequivocal. The compound, dSAM, was generated from SAM by the Escherichia coli enzyme in 87% yield, calculated on the assumption that dSAM had an extinction coefficient identical to that of SAM (21, 28). Low yields of dSAM via enzymatic decarboxylation of SAM, arise from the relatively high Km of SAM in the reaction, as well as feedback inhibition by dSAM. Thus, stoichiometry in the decarboxylation has been demonstrated only by coupling spermidine synthase and putrescine to the system to trap the aminopropyl of dSAM in spermidine and to compare the yield of this triamine to that of CO2 (12,14).A newly developed analytical system has been applied to the determination of picomole amounts of SAM and dSAM (9). We have determined the course of the SAM decarboxylase reaction and have established the equimolar production of dSAM and CO2 from SAM by t...

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SVS-1/SUSD2 is a novel gene, which inhibits growth and reverses