Antiretroviral activity of two polyisoprenylated acylphloroglucinols, 7-epi-nemorosone and plukenetione A, isolated from Caribbean propolis.

OBJECTIVES: Polyisoprenylated acylphloroglucinols have recently emerged as antitumoral agents. This study aims at elucidating the antiretroviral activity of two such compounds which were isolated from Caribbean propolis: 7-epi-nemorosone and plukenetione A, the structure of which is based on an adamantane moiety. Plukenetione A is for the first time shown to have antiretroviral activity. MATERIAL AND METHODS: The isolation of both small molecules was carried out using RP-HPLC. Their antiretroviral activity was studied based on lentiviral particles produced in HEK293T cells from the SIV-based vector VLDBH; their cytotoxicity was monitored by MTT proliferation assay. The antiviral activity of 7-epi-nemorosone was studied in CEMx174-SEAP infected with the HIV-1-strain pNL4.3wt. Reverse transcriptase inhibition was determined by a standard two-step RT-PCR using MMLV RT. RESULTS: 7-epi-nemorosone and plukenetione A were found to be potent antilentiviral agents in the employed system, inhibiting viral infection at concentrations below 1 µM/2 µM, respectively. Whereas 7-epi-nemorosone was not able to inhibit the reverse transcriptase in vitro (IC50 > 25 µM), plukenetione A effectively inhibited its enzymatic activity at an IC50 of 1.75 µM. CONCLUSIONS: Despite 7-epi-nemorosone and plukenetione A sharing some structural core elements, the mechanism of action involved in their antiretroviral activity seems to be different. We propose that 7-epi-nemorosone inhibits the viral replication by interrupting the Akt/PKB signaling cascade, as was demonstrated previously in various cell lines. Since plukenetione A effectively inhibits the enzymatic activity of MMLV reverse transcriptase at concentrations that show antilentiviral activity, we suggest that this small molecule acts by interfering with the enzyme's catalytic site.

Nemorosone blocks proliferation and induces apoptosis in leukemia cells.

OBJECTIVE: This work is aimed at characterizing nemorosone, isolated from Clusia rosea, as a potential antileukemic agent. In addition, we analyzed its influence on hematopoiesis in a mouse model. MATERIALS AND METHODS: The isolation of nemorosone was carried out employing the RP-HPLC (reversed phase high-performance liquid chromatography) technique. Cytotoxicity was assessed in human leukemia cell lines including parental and chemotherapy-refractory sublines based on the MTT compound. Its effects on the cell cycle were analyzed using FACS (fluorescence-activated cell sorting) and Western blot techniques. Studies on the drug-induced early apoptotic process were carried out by means of fluorescence microscopy. Major signal transducers and the enzymatic inhibition of immunoprecipitated Akt/PKB were detected by Western blot. Hematopoiesis was analyzed in NMRI nu/nu mice after chronic nemorosone treatment, measuring hematological parameters by conventional laboratory techniques. RESULTS: Nemorosone proved cytotoxic in both parental and chemoresistant leukemia cell lines with IC50 values between 2.10 and 3.10 mg/ml. No cross-resistances could be detected. Cell cycle studies showed apoptosis induction accompanied by an increase in the G0/G1 population in both cell lines studied, whereas a significant decrease in the S-phase was found in Jurkat cells. Nemorosone induced a down-regulation of cyclins A, B1, D1, and E as well as a dephosphorylation of cdc2. Major signal transduction elements such as ERK1/2 and p38 MAPK, as well as important oncoproteins such as c-Myb and BCR/ABL were also found down-regulated. The enzymatic activity of immunoprecipitated Akt/PKB was substantially inhibited in vitro. Moreover, subchronic nemorosone treatment induced reversible monocytosis and thrombocytosis in the mouse model examined. CONCLUSIONS: Here, we demonstrate for the first time that nemorosone exerts cytotoxicity in leukemia cells, partly by targeting the Akt/PKB signal transducer, affecting protein levels and cell cycle progression. Finally, in vivo studies suggest that nemorosone significantly affects hematopoiesis in mice.

Mucronulatol from Caribbean propolis exerts cytotoxic effects on human tumor cell lines.

OBJECTIVE: Mucronulatol is one of the most cytotoxic substances present in Caribbean propolis. This work aimed at initially characterizing the biological effects of mucronulatol in cancer cell lines comprehending both wildtype and resistant sublines. MATERIALS AND METHODS: An RP-HPLC technique was employed to separate and purify mucronulatol. IC(50) values were determined using the sulforhodamine B (SRB) proliferation assay. FACS-based cell cycle studies were carried out combining propidium iodide staining and 5-bromo-2'-deoxyuridine incorporation. Cell cycle regulator proteins were detected by Western blotting. The transcription of genes of interest was analyzed using RT-PCR. RESULTS: In MDR1-/MDR3+ cells, mucronulatol exhibited cytotoxicity in the range of 2.7 - 10.2 microg/ml, while no cytotoxic effects were observed in MDR1+ systems at up to 100 microg/ml. Cytometric studies revealed that mucronulatol promoted a global reduction in all cell cycle phases, with a remarkable increase of the apoptotic sub-G1 population. Immunoblotting showed that mucronulatol induced an up-regulation of p21(Cip1) and p27(Kip1) while down-regulating cyclin E and CDK4 in a drug concentration-dependent manner. No effect on topoisomerase I was observed, while we detected an altered expression of topoisomerases II-I+/-/I(2). RT-PCR studies showed that 2-fold the IC(50) in HCT8 colon carcinoma cells was sufficient for altering the expression pattern of genes in this cell line, including topoisomerase I, thymidilate synthase, EGF receptor and c-myc, amongst others. CONCLUSION: Here, we demonstrate for the first time that mucronulatol exerts cytotoxicity in cancer cell lines by targeting the control of cell cycle progression, indicating that the mechanism of action of this compound involves interference with the cell cycle machinery.

Cytotoxic activity of nemorosone in neuroblastoma cells.

Neuroblastoma is the second most common solid tumour during childhood, characterized by rapid disease progression. Most children with metastasized neuroblastoma die despite intensive chemotherapy due to an intrinsic or acquired chemotherapy resistance. Thus, new therapeutic strategies are urgently needed. Here, we demonstrate that the novel compound nemorosone isolated from alcoholic extracts of Clusia rosea resins by reverse phase high pressure liquid chromatography (RP-HPLC) exerts cytotoxic activity in neuroblas-toma cell lines both parental and their clones selected for resistance against adriamycin, cisplatin, etoposide or 5-fluorouracil. Cell cycle studies revealed that nemorosone induces an accumulation in G0/G1- with a reduction in S-phase population combined with a robust up-regulation of p21Cip1. Furthermore, a dose-dependent apoptotic DNA laddering accompanied by an activation of caspase-3 activity was detected. Nemorosone induced a significant dephosphorylation of ERK1/2 in LAN-1 parental cells probably by the inhibition of its upstream kinase MEK1/2. No significant modulation of signal transducers JNK, p38 MAPK and Akt/PKB was detected. The enzymatic activity of immunoprecipitated Akt/PKB was strongly inhibited in vitro, suggesting that nemorosone exerts its anti-proliferative activity at least in part by targeting Akt/PKB in the cell lines studied. In addition, a synergistic effect with Raf-1 inhibitor BAY 43-9006 was found. Finally, nemorosone induced a considerable down-regulation of N-myc protein levels in parental LAN-1 and an etoposide resistant sub-line at the same drug-concentrations.

Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells.

Overexpression of the detoxifying enzyme cytidine deaminase (CDD) renders normal and leukemic hematopoietic cells resistant to cytarabine (1-beta-D-arabinofuranosylcytosine), and studies on murine cells have suggested transgenic CDD overexpression as a way to reduce the substantial myelotoxicity induced by the deoxycytidine analogs cytarabine and gemcitabine (2',2'-difluorodeoxycytidine). We now have investigated CDD (over-)expression in the human hematopoietic system. Retroviral gene transfer significantly increased the resistance of CDD-transduced cord blood and peripheral blood-derived progenitor cells for doses ranging from 20-100 nM cytarabine and 8-10 nM gemcitabine. Protection was observed for progenitors of erythroid as well as myeloid differentiation, though the degree of protection varied for individual drugs. In addition, significant selection of CDD-transduced cells was obtained after a 4-day culture in 30-100 nM cytarabine. Thus, our data demonstrate that overexpression of CDD cDNA results in significant protection of human progenitors from cytarabine- as well as gemcitabine-induced toxicity, and allows in vitro selection of transduced cells. This strongly argues for a potential therapeutic role of CDD gene transfer in conjunction with dose-intensive cytarabine- or gemcitabine-containing chemotherapy regimen.

Gene therapy and the skin.

Significant progress has been made during the past decade in corrective gene therapy of the skin. This includes advances in vector technology, targeted gene expression, gene replacement, gene correction, and the availability of appropriate animal models for a variety of candidate diseases. While non-viral integration of large genes such as essential basement membrane proteins has been mastered, new challenges such as the control of immune responses lie ahead of the research community. Among the first skin diseases, patients with junctional epidermolysis bullosa (JEB) and xeroderma pigmentosum (XP) will enter clinical trials.

Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

Identification of the full-length huntingtin- interacting protein p231HBP/HYPB as a DNA-binding factor.

Neurodegeneration in Huntington's disease (HD) is associated with an elongated glutamine tract in the widely expressed huntingtin protein. Although the pathogenic mechanisms are still unknown, the distinct physical properties of mutant huntingtin in the brain suggest that other factors including huntingtin-interacting proteins might play a specific role. We have previously identified a DNA-binding motif in the proximal E1A promoter of adenovirus serotype 12 as responsible for E1A autoregulation. Here, we identified the p231HBP protein as a DNA-binding factor, the C-terminal portion of which has recently been characterized as the huntingtin-interacting protein HYPB of unknown function. We have determined the full-length cDNA sequence, identified several domains supporting its gene regulatory functions, and mapped the HBP231 gene to chromosome 3p21.2-p21.3. Our results provide an interesting molecular link between huntingtin and a DNA-binding factor, implicating that this interaction might result in the alteration of cellular gene expression involved in HD pathogenesis.

Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene.

PURPOSE: Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34+ cells. METHODS: The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFbeta1-EGFP) or EGFP and a downstream (vector SFbeta1-EGFP-IRES) or upstream (vector SFbeta1-IRES-EGFP) internal ribosomal entry site. RESULTS: For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 +/- 0.4), followed by N2A-PGK-EGFP (26.3 +/- 1.8 MFI), and N2A-TK-EGFP (4.8 +/- 0.1 MFI). Expression from the SFbeta1-EGFP vector (82.6 +/- 6.7 MFI) and the SFbeta1-EGFP-IRES vector (102.8 +/- 6.2 MFI) was higher than from SFbeta1-IRES-EGFP vector (15.5 +/- 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFbeta1-EGFP vector still being four- to fivefold more active than the internal promoters tested. CONCLUSION: The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration.

Differential susceptibility of renal carcinoma cell lines to tumor suppression by exogenous Fhit expression.

Hemizygous deletions of the fragile histidine triad (FHIT) gene at human chromosome band 3p14.2 and down-regulation of its gene product are found in the majority of renal cell carcinomas (RCCs). Functional tumor suppressive activity of Fhit in renal cancer cells previously was observed in RCC cell line RC48, which lacks endogenous Fhit expression. To further investigate the potential role of FHIT as a tumor suppressor gene in RCC, we transfected FHIT cDNA expression constructs into RCC cell lines RCC-1 and SN12C, which show low-level expression of endogenous Fhit and reveal an intact von Hippel-Lindau (VHL) gene. Stable transfectants of both cell lines showed no alterations of cell morphology, proliferation kinetics, or cell cycle parameters in vitro. The FHIT gene transfer rate, however, was significantly lower in RCC-1 cells compared with SN12C cells, suggesting a selection against exogenous Fhit expression. In addition, in nude mouse assays, a significant delay of tumor formation was observed for FHIT-transfected RCC-1 cell lines, with outgrowing tumors demonstrating loss of Fhit expression in the majority of cells. In contrast, tumorigenicity of FHIT-transfected SN12C cell clones was not suppressed, despite stable transgene expression. In conclusion, our results demonstrate a selective tumor suppressive activity of Fhit in RCC cells in vivo and suggest that the susceptibility to suppression is not restricted to cancer cells with complete loss of Fhit expression.

The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma.

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.

Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments.

BACKGROUND: Retroviral transduction in the presence of fibronectin (FN) fragments has proven an efficient and clinically-applicable procedure for gene transfer into hematopoietic cells. So far, FN-based transduction protocols have been optimized primarily for transduction of stem cells, whereas for several therapeutic applications transduction of clonogenic progenitors (CFU) may be sufficient. METHODS: Transduction protocols for CFU were optimized by evaluating the effect of growth factors, timing of retroviral transduction, CD34-selection and heparin, using a neomycin-phosphotransferase (neo(R))-expressing retroviral vector. RESULTS: The presence of multiple growth factors during prestimulation and transduction, including the differentiating cytokines G-CSF or GM-CSF, substantially enhanced transduction of CFU. Best results were achieved when 24 h of prestimulation were followed by a 24-48 h transduction period in the presence of the CH-296 FN-fragment and IL-3, IL-11, SCF, erythropoietin (EPO), and GM-CSF. With this proto-col we observed highly efficient transduction of BM-derived CFU (90.7 +/- 8.8 % G 418-resistant colonies), even with retrovirus preparations of moderate infectious titer (5 x 10(4) - 2 x 10(5) CFU/mL). The number of CFU increased on average 2.6-fold (range 1.5-3.8) during the transduction procedure. Selection of CD34(+) cells prior to transduction did not improve transduction efficiency. Heparin, even in concentrations as low as 2.0 microg/mL, significantly inhibited transduction of CFU on FN-fragments. DISCUSSION: An optimized protocol for retroviral gene transfer into human clonogenic progenitor cells that allows highly efficient transduction, even with moderate titer retroviral vectors, is presented.

Novel tumor suppressor locus in human chromosome region 3p14.2.

BACKGROUND: Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. METHODS: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. RESULTS: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. CONCLUSION: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.

Minimal deletion of 3p13-->14.2 associated with immortalization of human uroepithelial cells.

Immortalization and tumorigenic transformation of many human cell types, including human uroepithelial cells (HUCs), are frequently associated with loss of genetic material from the short arm of chromosome 3 (3p). In addition, losses of 3p have been observed in many human cancers including renal cell carcinoma, lung cancer, breast cancer, and bladder cancer. Genetic studies suggest that there are at least two regions on 3p in which tumor suppressor genes might be located, but the precise location of these genes is not known. We studied chromosome 3 losses that were specifically associated with immortalization of five independent human papilloma virus 16 (HPV16) E6- or E7-transformed HUCs. Cytogenetic analysis showed that the smallest common region of deletion was 3p14.1-->14.2. Fluorescence in situ hybridization using a 3p13-->14-specific yeast artificial chromosome (YAC) contig showed the precise localization of the breakpoints to be in 3p13 and 3p14.2, thus defining the smallest common overlap of 3p deletions in HPV16 E6- or E7-immortalized HUCs. These results suggest the presence in this region of genes involved in the control of senescence in vitro and possibly tumorigenesis in vivo.

Yeast artificial chromosome transfer into human renal carcinoma cells by spheroplast fusion.

Successful transfer of yeast artificial chromosomes (YACs) into human cells has been described in only a single study. We here report on the evaluation of YAC transfer strategies into a human renal cell carcinoma cell line by yeast spheroplast fusion and cationic lipids. While the latter approach proved inefficient, significant numbers of clones containing both vector arms were obtained by spheroplast fusion. FISH analyses on such clones revealed the presence of YAC integration and the co-localization of both vector arms with insert sequences. These data demonstrate that under certain experimental conditions efficient YAC transfer into human cells by spheroplast fusion is possible and may be useful for the cloning of human disease-related genes.

Identification of novel 'expressed sequence tags' within the FHIT gene locus in human chromosome region 3p14.2.

Losses of genetic material within human chromosome regions (HCR) 3p12-p14 and 3p21-p22 are observed in various neoplasias, suggesting tumor suppressor gene (TSG) loci within these regions. HCR 3p14 is particularly interesting as it contains the t(3;8) translocation breakpoint of a hereditary renal cell carcinoma, the FRA3B fragile site, and DNA markers deleted in several types of human cancer. We here report on the identification of five novel 'expressed sequence tags' (ESTs) within 3p14.2 which map proximal to exon 9 of the candidate TSG, FHIT. These ESTs may be valuable for elucidation of the supposed TSG content in 3p14.2.

Genetic instability of 3p12-p21-specific microsatellite sequences in renal cell carcinoma.

OBJECTIVE: To determine the role of structural alterations of human chromosome region 3p12-p21 in the possible inactivation of one or more tumour-suppressor genes in the pathogenesis of renal cell carcinoma (RCC), lung cancer and other neoplasms. MATERIALS AND METHODS: As microsatellite instability (MI), in particular MI with loss of heterozygosity (LOH), may indicate putative tumour-suppressor gene loci, 20 kidney tumours, including 14 clear cell carcinomas and six non-clear cell neoplasms, were investigated with 10 polymorphic simple sequence-repeat markers spanning 3p12-p21. Six of these markers map to the region of deletion flanked by markers D3S1285 and D3S1295 bracketing the t(3;8) translocation break-point in 3p14.2 of hereditary RCC. RESULTS: Twelve of 14 clear cell RCCs displayed MI for at least one locus, as opposed to none of the non-clear cell tumours (P = 0.001). Locus D3S1274 in 3p13 located in the region deleted in lung cancer line U2020 and loci D3S1313 and D3S1300 in 3p14.3 characterized common regions of instability and LOH. Two patients with RCC who also had lung cancer and colon cancer, respectively, showed LOH at D3S1313 or D3S1300 as the only alterations of their kidney tumours. CONCLUSIONS: These results suggest that human chromosome region 3p14.3 distal to the hereditary t(3;8) translocation breakpoint and the region deleted in the U2020 lung cancer cell line might be involved in the tumorigenesis or progression of clear cell RCC.