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T Tamaoki

Publications and source records attributed to T Tamaoki.

At least 19 recordsLinked to original sources
PubMed2000

Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G(1) phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex.

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimeric bcr-abl gene giving rise to a p210(Bcr-Abl) protein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G(1) phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210(Bcr-Abl), Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle-dependent kinases 4 and 6 and up-regulated cell cycle-dependent kinase inhibitor p27(Kip1) protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210(Bcr-Abl) formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210(Bcr-Abl) from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210(Bcr-Abl).

Animals↗
PubMed2000

The mouse ZFH-4 protein contains four homeodomains and twenty-two zinc fingers.

We isolated the mouse zfh-4 cDNA which is 12 kb long and capable of encoding a 3,550-amino acid protein containing four homeodomains and 22 zinc fingers including two pseudo zinc finger motifs. The mouse ZFH-4 is 51% homologous to the mouse ATBF1 and 23% to the Drosophila ZFH-2. The homeodomain and zinc finger regions are highly conserved between ZFH-4 and ATBF1 except that one zinc finger is missing in ZFH-4. Analysis of partial genomic sequences showed that the mouse zfh-4 and ATBF1 genes are similar in exon-intron organization. RT-PCR analysis of zfh-4 transcripts in adult mouse tissues showed that zfh-4 expression was low but reproducibly detectable in brain, heart, lung and muscle. In these mouse tissues, ATBF1 transcripts were poorly amplified by PCR under the conditions where zfh-4 transcripts were amplified, suggesting that the expression of zfh-4 mRNA is higher than that of ATBF1 mRNA. Other comparative analysis suggests functional similarities and dissimilarities between ZFH-4 and ATBF1.

Amino Acid Motifs↗
PubMed2000

Chemoprotective effects of KF41399, a derivative of carbazole compounds, on nimustine-induced thrombocytopenia.

We examined the chemoprotective effects of KF41399, a novel derivative of carbazole compounds, on severe thrombocytopenia induced by nimustine (ACNU, 45 mg/kg administered for 2 consecutive days intravenously) in mice. Administration schedule studies revealed that pretreatment of mice with KF41399 was necessary to improve thrombocytopenia. Oral administration of KF41399 ameliorated thrombocytopenia induced by ACNU and accelerated the rate of platelet recovery in a dose-dependent fashion. In addition, KF41399 pretreatment improved the decrease in body weight and spleen weight and in the colony-forming activity of bone marrow mononuclear cells (MNC). Oral administration of KF41399 to normal mice induced G(0)/G(1)-phase accumulation of MNC as well as hematopoietic progenitor cells (lineage negative cells [Lin(-)]) and reduced the colony-forming activity of MNC. In Lin(-) cells derived from KF41399-treated mice, up-regulation of Bcl-2 and down-regulation of cyclin E and cyclin A proteins were observed. In the same cells, a decrease in the phosphorylated form of Rb protein and an increase in the p130 protein were observed without changes in the protein level of cell cycle-dependent kinase 2 (Cdk2), Cdk4, and Cdk6. More important, KF41399 did not affect the antitumor activity of ACNU against mouse Sarcoma180 and human lung cancer LC-6. However, 25-mg/kg KF41399 treatment reduced the antitumor activity of ACNU against human lung cancer Lu-65, and 5 mg/kg KF41399 caused a slight reduction of the antitumor activity of ACNU without inducing thrombocytopenia. These results suggest that KF41399 might be useful as a chemoprotective agent to improve chemotherapy-induced thrombocytopenia and types of other toxicity. (Blood. 2000;95:3771-3780)

Adenocarcinoma↗
PubMed2000

Expression and role of p27(kip1) in neuronal differentiation of embryonal carcinoma cells.

We examined the expression and the regulation of p21(waf1) and p27(kip1) cdk inhibitors in P19 mouse embryonal carcinoma (EC) cells following treatment with all-trans retinoic acid (ATRA) to induce neuronal differentiation. The levels of p27 mRNA and protein increased within 24 h of treatment with ATRA, reaching a plateau 4-5 days later prior to neurite formation. In contrast, levels of p21 expression remained low until after neurites were extensively formed. Induction of muscle differentiation from P19 cells by treatment with dimethyl sulfoxide caused only transient increases in p27 levels. In a mutant P19 cell line, RAC65, treatment with ATRA induced neither p27 accumulation nor neuronal differentiation, but p21 mRNA expression increased markedly. In contrast, treatment of RAC65 cells with 9-cis retinoic acid induced both p27 expression and neuronal differentiation. Correlation between p27 expression and neuronal differentiation was also observed in NT2/D1 human EC cells. Luciferase reporter assays showed that p27 promoter activity increased in ATRA-treated cells, consistent with the elevation of p27 mRNA levels. Arrest of neuronal differentiation of P19 cells by okadaic acid resulted in inhibition of p27 expression, whereas p21 mRNA expression was greatly enhanced. Conversely, inhibition of p27 expression by antisense p27 oligonucleotides resulted in blockade of neuronal differentiation. Taken together, these results strongly suggest that the expression of p27 is indispensable for neuronal differentiation of EC cells.

Animals↗
PubMed2000

UCN-01 selectively enhances mitomycin C cytotoxicity in p53 defective cells which is mediated through S and/or G(2) checkpoint abrogation.

We have previously reported that UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor that is under clinical trials as an anti-cancer agent in the USA and Japan, enhanced the anti-tumor activity of mitomycin C (MMC) in vitro and in vivo. Subsequent studies from other laboratories revealed that UCN-01 could selectively enhance cytotoxicity of DNA damaging agents in p53 defective cells and that this was mediated by abrogation of S and /or G(2) arrest by UCN-01. In this study, we report that UCN-01 selectively enhances the cytotoxicity of MMC in human p53 mutant cell lines. In contrast, UCN-01 showed little, if any, effect on MMC cytotoxicity in human p53 wild-type cell lines. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) assay revealed that the combination of MMC with UCN-01 increased DNA breaks consistent with apoptosis in p53 defective A431 epidermoid carcinoma cells. In p53 wild-type MCF-7 breast carcinoma cells, the cyclin-dependent kinase inhibitor protein p21/WAF1 was markedly induced after the treatment with MMC alone, although this response was significantly delayed from the time of MMC treatment. Detailed cell-cycle studies revealed that UCN-01 abrogated S and G(2) phase accumulation induced by MMC in p53 defective cells and to a lesser extent in p53 wild-type cell lines. The abrogation of arrest in p53 wild-type cells was observed prior to significant induction of the p53 response. Since MMC was less effective against p53 defective cell lines than against p53 wild-type cell lines and UCN-01 selectively enhanced MMC cytotoxicity in p53 defective cell lines, UCN-01 may provide a new modality of MMC-based cancer chemotherapy, particularly in p53 defective cancer patients.

Alkaloids↗
PubMed2000

AT motif binding factor 1-A (ATBF1-A) negatively regulates transcription of the aminopeptidase N gene in the crypt-villus axis of small intestine.

This is the first study to demonstrate that the AT motif binding factor 1-A (ATBF1-A) is expressed in the crypts and the bases of villi of the small intestine and negatively regulates transcription of brush-border enzyme gene, aminopeptidase-N (APN). In situ hybridization visualized a limited ATBF1-A mRNA expression in the crypts and the bases of villi. Transient transfection and dual luciferase-reporter assay demonstrated that ATBF1-A suppressed the activity of APN promoter, but did not that of AT motif deleted promoter. These results imply that ATBF1-A inhibits the transcription of APN gene through its direct binding to the AT motif element. Furthermore, butyrate-induced differentiation of Caco-2 cells, retaining the enterocytic phenotypes such as a villus structure and the expression of brush-border enzymes, leads to a reduced expression of ATBF1-A mRNA. We proposed that ATBF1-A regulating APN gene expression in the crypt-villus axis of the small intestine is a landmark of enterocyte differentiation and maturation.

Animals↗
PubMed2000

Human alpha-fetoprotein transcriptional regulatory sequences. Application to gene therapy.

The AFP regulatory sequences are among the best known tumor-specific transcriptional regulators. A number of groups have demonstrated that a variety of genes can be expressed in an HCC-specific manner under the control of the AFP regulatory sequences in vitro and in vivo. It would appear that, with the development of a suitable delivery system, HCC-directed gene therapy using the AFP regulatory sequences holds a promising future.

Animals↗
PubMed2000

Synthesis and antitumor activity of duocarmycin derivatives: modification at C-8 position of A-ring pyrrole compounds bearing the simplified DNA-binding groups.

A series of the 8-O-substituted A-ring pyrrole derivatives of duocarmycin bearing the simplified DNA-binding moieties such as cinnamoyl or heteroarylacryloyl groups were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. In addition, the stability of the 8-O-substituted analogues in aqueous solution and the conversion to their active form (cyclopropane compound) from the 8-O-substituted analogues in mice or human serum were examined. The 8-O-substituted A-ring pyrrole derivatives bearing the simplified DNA-binding moieties showed remarkably potent in vivo antitumor activity and low peripheral blood toxicity compared with the 8-O-substituted A-ring pyrrole derivatives having the trimethoxyindole skeleton in segment-B (Seg-B), which were equal to 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4'-methoxycinnamates and 4'-methoxy-beta-heteroarylacrylates. Moreover, among 8-O-substituted analogues, several compounds can be chemically or enzymatically converted to their active form in human serum. This result indicated that new 8-O-substituted derivatives were different prodrugs from KW-2189 and 8-O-substituted analogues being the same type of prodrug as KW-2189.

Animals↗
PubMed1999

Hepatoma-specific gene therapy through retrovirus-mediated and targeted gene transfer using an adenovirus carrying the ecotropic receptor gene.

Tissue-specific transcriptional regulatory sequences have been inserted in retrovirus vectors for therapeutic gene expression in cancer gene therapy. However, the transcriptional activity of these sequences is generally low, and the proviral DNA integration appears to increase the possibility of genomic DNA alteration in nontarget cells. Therefore, retrovirus-mediated targeted gene transduction into human hepatoma cells was evaluated using transient expression of an ecotropic receptor gene, mouse cationic amino acid transporter-1 (MCAT-1). Two recombinant adenoviruses, AxCAMCAT and AxAFMCAT, carrying the MCAT-1 gene under the control of the CAG and human alpha-fetoprotein (AFP) promoter, respectively, were generated. The preinfection with AxCAMCAT allowed highly efficient ecotropic retroviral infection of human cells. In addition, after AxAFMCAT infection, retroviral infection occurred only in AFP-producing hepatoma cells, resulting in selective cytotoxicity induced by the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system. Transient expression of the MCAT-1 gene under the control of the AFP promoter permits ecotropic retrovirus-mediated gene transduction specifically in AFP-producing human hepatoma cells, resulting in selective induction of the suicide killing effect while the therapeutic gene is driven from ubiquitously expressed promoters.

3T3 Cells↗
PubMed1999

Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-Hydroxystaurosporine).

7-Hydroxystaurosporine (UCN-01) is a protein kinase inhibitor that is under development as an anticancer agent in the United States and Japan. Long-term exposure of human A549 non-small cell lung cancer cells to UCN-01 furnished cells (A549/UCN) with acquired resistance against UCN-01. In this study, the sensitivity of these cells toward the growth-arresting properties of certain conventional cytotoxic agents was explored. Cells were not cross-resistant against adriamycin, Taxol, staurosporine, and UCN-02, but they displayed 14- and 4.4-fold resistance against cisplatin and mitomycin C, respectively. Previous studies on the mechanism(s) of action of UCN-01 suggest that induction of apoptosis and G1 phase accumulation are important for its anticancer activity; therefore, we compared induction of apoptosis and cell cycle distribution caused by UCN-01 in wild-type A549 and A549/UCN cells using flow cytometry. UCN-01 (0.4 microM) induced apoptosis (62% terminal deoxynucleotidyl transferase-mediated nick end labeling-positive cells) in A549 cells, but not in A549/UCN cells. The percentages of cells that accumulated in G1 when exposed to UCN-01 (0.4 microM) were 22% in A549 cells and 67% in A549/UCN cells. These results suggest that acquired resistance of cancer cells against UCN-01 is characterized by attenuation of apoptosis induction associated with reinforcement of the G1 checkpoint and that apoptosis regulation is drastically altered in A549/UCN cells as compared with A549 cells. Cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 in A549/UCN cells were up-regulated, which was accompanied by overexpression of G1 cyclins D1 and E, but UCN-01 hardly affected levels of these proteins. In contrast, cyclin A, cyclin B1, retinoblastoma, and CDK2 proteins were apparently down-regulated, without changes in CDK4/6. UCN-01 hardly affected the expression level of cyclin B1 and induced dephosphorylation of retinoblastoma in both cell types. UCN-01 induced down-regulation of cyclin A level and CDK2 activity accompanied with its dephosphorylation in A549/UCN cells, but not in A549 cells. The antiapoptotic protein bcl-2 was apparently up-regulated in A549/UCN cells, however, bcl-xL, another antiapoptotic protein, was down-regulated, without changes in bak and bax. Taken together, these results are consistent with the notion that induction of apoptosis and block of cell cycle in G1 are important determinants of the sensitivity of cancer cells to UCN-01 and suggest that inhibition of CDK2 activity accompanied by its dephosphorylation and decrease of expression level of cyclin A might play an important role in the G1 phase accumulation induced by UCN-01.

Alkaloids↗
PubMed1999

Synthesis and antitumor activity of duocarmycin derivatives: modification of segment-A of A-ring pyrrole compounds.

A series of 3-substituted A-ring pyrrole compounds of duocarmycin were synthesized and evaluated for in vitro anticellular activity against HeLa S(3) cells and in vivo antitumor activity against murine sarcoma 180 in mice. These compounds were evaluated on the peripheral blood toxicity and delayed lethal toxicity. Further, to expand our investigation of their peripheral blood toxicity, the toxicity to bone marrow cells (CFU-GM, CFU-Meg) was investigated. Among 3-substituted A-ring pyrrole compounds of duocarmycin bearing a 5',6',7'-trimethoxy-2'-indolecarboxyl group as segment-B (Seg-B), several analogues showed remarkably potent antitumor activity with low peripheral blood toxicity. The 3-formyl compound 12h, one of such analogues, showed stronger antitumor activity with lower toxicity to bone marrow cells compared to DU-86 (2a), an active metabolite of KW-2189 (2b). However, compound 12h caused delayed death. On the other hand, the 3-bromo compound 15f, one of the 3-substituted A-ring pyrrole derivatives bearing a 4'-methoxycinnamoyl group as Seg-B, showed the most potent antitumor activity among the 4'-methoxycinnamate analogues with low toxicity to bone marrow cells. Furthermore, compound 15f did not cause delayed death similarly to 2d. These results would indicate the importance of the C-3 substituents of A-ring pyrrole duocarmycin derivatives for exhibiting antitumor activity and decreasing toxicity.

Animals↗
PubMed1999

KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules.

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.

Animals↗
PubMed1999

Antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine, a novel deoxyadenosine analog, against human colon tumor xenografts by oral administration.

UNLABELLED: 2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine (Cl-F-araA) is a novel deoxyadenosine analog, which inhibits DNA synthesis by inhibiting DNA polymerase alpha and ribonucleotide reductase. Cl-F-araA shows potent antiproliferative activity against several leukemic cell lines including those of human origin and is also effective against murine solid tumors, in particular being curative against colon tumors. PURPOSE: We therefore decided to investigate whether Cl-F-araA is effective against human colon tumors, in particular by oral administration, since it has improved stability compared with other deoxyadenosine analogs. METHODS: Antiproliferative activity in vitro was determined from cell counts. Subcutaneously inoculated xenograft models and a liver micrometastases model were used for assessment of antitumor activity in vivo. RESULTS: Cl-F-araA showed potent antiproliferative activity against four human colon tumor cell lines (HCT116, HT-29, DLD-1, WiDr), with a 50% growth-inhibitory concentration (IC50) of 0.26 microM with a 72-h exposure. This activity was greater than those of fludarabine desphosphate and cladribine, other deoxyadenosine analogs, which showed IC50 values of 19 microM and 0.35 microM, respectively. Cl-F-araA showed potent antitumor activity against four human colon tumor xenograft models (HT-29, WiDr, Co-3, COLO-320DM) in a 5-day daily administration schedule, which was shown to be the most effective of three administration regimens tested (single, twice-weekly, 5-day daily). In particular, oral administration showed significantly superior activity, with a regressive or cytostatic growth curve, compared with intravenous administration. In addition, Cl-F-araA was effective at only one-sixteenth of the maximum dose tested in a 10-day daily administration schedule. Therapeutic efficiency seemed to increase in proportion to the frequency of administration. Cl-F-araA also decreased liver micrometastases created by intrasplenic injection of human colon tumor cells, leading to complete suppression at the maximum dose tested. CONCLUSIONS: These results suggest that Cl-F-araA might be clinically effective against human colon cancers using a daily oral administration schedule.

Adenine Nucleotides↗
PubMed1999

Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole.

UCN-01 (7-hydroxystaurosporine) and CGP 41 251 (4'-N-benzoyl staurosporine), both of which were discovered as protein kinase C selective inhibitors, have entered in phase 1 clinical trials as anti-cancer drugs. In this study, we have directly compared the effects of these drugs as well as staurosporine (STP) on cell cycle progression of A431 human epidermoid carcinoma cells synchronized at M phase by treatment with nocodazole. The nocodazole-synchronized cells progressed from M to G1 phase in the absence of the drug, which was accompanied by a decrease of cyclin B1 protein expression, disappearance of the complex formation of CDC2 with cyclin B1 and reduction of the kinase activity. Treatments of the M phase cells with UCN-01, STP and CGP 41 251 at 80% growth-inhibitory concentrations (IC80S) resulted in specific G1 block, G2M block and polyploidy, respectively. Decreases of cyclin B1 protein expression was partially prevented by treatments with STP and CGP 41 251 but not with UCN-01 at IC80S. Reductions of active complex and kinase activity of CDC2/cyclin B1 were also observed in the presence of the three drugs. In addition, augmentation of CDC2 protein tyrosine phosphorylation was induced only when the cells were treated with STP. These observations demonstrated that higher concentrations of UCN-01, STP and CGP 41 251 showed different effects on cell cycle progression as well as CDC2/cyclin B1 regulation in A431 cells synchronized at M phase. The data suggest that UCN-01 and CGP 41 251 may act at quite different points on the cell cycle.

Alkaloids↗
PubMed1999

Antitumor activity of KF22678, a novel thioester derivative of leinamycin.

KF22678, a novel thioester derivative of leinamycin with the 1-oxo-1,2-dithiolane-3-one moiety, was examined for anti-tumor activity, toxicity in mice and activation mechanism. KF22678 showed a broad antitumor spectrum against human carcinoma xenografts (lung, colon, ovary and prostate). The efficacy of KF22678 was significantly higher than that of cisplatin. KF22678 exhibited low cross-resistance against various drug-resistant cell lines of MDR1 or MRP overexpressing human tumors, and, in addition, exhibited more potent antitumor activity in vivo than ADM against A2780/ADM and KB/MRP xenograft. DL-Buthionine sulfoximine (BSO) pretreatment significantly reduced intracellular glutathione (GSH) level in human lung carcinoma A549 cells, leading to decrease in the cytotoxicity of KF22678, whereas the cytotoxicity of melphalan was augmented by BSO pretreatment. DNA single-strand breaks (SSB) were observed in A549 cells treated with KF22678 and bleomycin. DNA SSB induced by KF22678 was greatly reduced in the presence of BSO in the cells, whereas DNA SSB induced by bleomycin was not. In addition, the antitumor activity of KF22678 against BSO-pretreated human lung carcinoma PC-9 tumor was significantly decreased. These results suggest that the activation of KF22678 by intracellular GSH might be important for DNA SSB and antitumor activity in vitro and in vivo.

Animals↗
PubMed1999

G1-checkpoint function including a cyclin-dependent kinase 2 regulatory pathway as potential determinant of 7-hydroxystaurosporine (UCN-01)-induced apoptosis and G1-phase accumulation.

7-Hydroxystaurosporine (UCN-01), which was originally identified as a protein kinase C selective inhibitor, is currently in clinical trials as an anti-cancer drug. We previously showed that UCN-01 induced preferential G1-phase accumulation in tumor cells and this effect was associated with the retinoblastoma (Rb) protein and its regulatory factors, such as cyclin-dependent kinase 2 (CDK2) and CDK inhibitors p21Cip1/WAF1 and p27Kip1. We demonstrate here that G1-phase accumulation was induced by UCN-01 in Rb-proficient cell lines (WiDr and HCT116 human colon carcinomas and WI-38 human lung fibroblast), and it was accompanied by dephosphorylation of Rb. In addition, UCN-01-induced G1-phase accumulation was also demonstrated in a Rb-defective cell line (Saos-2 human osteosarcoma), but not in a simian virus 40 (SV40)-transformed cell line (WI-38 VA13). Apoptosis was induced by UCN-01 in the two Rb-deficient cell lines, but not in the other Rb-proficient cell lines. These observations suggest that G1-checkpoint function might be important for cell survival during UCN-01 treatment. In addition, there may be a UCN-01-responsive factor in the G1-checkpoint machinery other than Rb which is targeted by SV40. Further studies revealed a correlation between UCN-01-induced G1-phase accumulation and reduction of cellular CDK2 kinase activity. This reduction was strictly dependent on down-regulation of the Thr160-phosphorylated form of CDK2 protein, and coincided in part with up-regulation of p27Kip1, but it was independent of the level of the p21Cip1/WAF1 protein. These results suggest that G1-checkpoint function, including a CDK2-regulatory pathway, may be a significant determinant of the sensitivity of tumor cells to UCN-01.

Alkaloids↗
PubMed1998

A novel strategy of cell targeting based on tissue-specific expression of the ecotropic retrovirus receptor gene.

Gene transfer into specific tissues or cell types is a key technique in the development of gene therapy. Modification of vector particles such that they selectively bind to the target cells has been attempted, but the limitation of this approach is the low transduction efficiency. Here, we show that a two-step gene transfer system can be used for efficient cell targeting. With this strategy, and using a high-titer adenoviral vector containing a tissue-specific promoter, we have engineered a system in which only target cells become susceptible to retrovirus-mediated transduction. In a model experiment, we constructed an adenoviral vector (Ad.AFPEcoRec) containing the ecotropic retrovirus receptor (EcoRec) gene under the control of the alpha-fetoprotein (AFP) promoter. A binding assay showed that after transduction with AD.AFPEcoRec, EcoRec molecules were efficiently expressed in AFP+HepG2 cells, but not in AFP-HeLa and AFP-HLE cells. The EcoRec-expressing HepG2 cells could be stably transduced with ecotropic retroviral vectors, whereas HeLa and HLE cells remained highly resistant to retrovirus-mediated gene transfer. The apparent titer on HepG2 cells was greater than 2 x 10(5) CFU/ml. Because various tissue-specific promoter/enhancer elements are available, the two-step system could be used as a general strategy for both ex vivo and in vivo targeted gene transfer.

3T3 Cells↗