Involvement of ligninolytic enzymes in degradation of wheat straw by Trametes trogii.

AIMS: Wheat straw is generated in billions of tons around the world every year and has not been fully used. This study sought to evaluate the delignification capacity and enzyme production of Trametes trogii MT strain and to clarify the changes of structure and chemical composition of wheat straw during the decay process. METHODS AND RESULTS: The results obtained revealed that the T. trogii MT strain has the ability to degrade lignin, cellulose as well as hemicellulose of wheat straw simultaneously. The strain can produce high activities of laccase, manganese peroxidase, xylanase, carboxymethylcellulase and feruloyl esterase but no lignin peroxidases during the decay process of a 60-day incubation period on wheat straw. Scanning electron microscopy observation and infrared spectroscopy analysis showed the lignin and carbohydrate of wheat straw were degraded with no obvious different levels. The low molecular mass fractions collected from the culture of the MT strains grown in wheat straw powder liquid medium showed high Fe(3+) chelating, reducing capacity and hydroxyl radical and hydrogen peroxide generation. CONCLUSIONS: Trametes trogii MT has a complex mechanism to degrade lignocellulose, in addition to the extracellular enzymatic systems, and has great potential as an attractive micro-organism used for the biological degradation of waste straws. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the dynamic changes of the ligninolytic enzymes of T. trogii MT during the degradation of wheat straw, and suggested that the decay patterns of wheat straw by T. trogii MT had some simultaneous type characteristics.

Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis.

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.

Characterization of the ETO and AML1-ETO proteins involved in 8;21 translocation in acute myelogenous leukemia.

The AML1 and ETO genes are disrupted by the nonrandom chromosomal translocation t(8;21) in acute myelogenous leukemia (AML). While the AML1 gene encodes a transcription factor indispensable for definitive hematopoiesis, the biological function of ETO is unknown. To understand the role of ETO and AML1-ETO in the pathogenesis of AML, the full length cDNAs of ETO and AML1-ETO were cloned and antibodies against AML1 and ETO proteins have been developed in our laboratory. Western blot analysis showed that ETO and AML1-ETO were identified as 70 kDa and 94 kDa proteins, respectively, and that both proteins, like AML1, were associated with the nuclear matrix. To examine whether the t(8;21)-positive AMLs expressed a 94-kDa AML1-ETO, protein fractions isolated from leukemia blasts of 10 patients with t(8;21)-positive AML and the Kasumi-1 cells were analyzed by Western blotting. The 94 kDa AML1-ETO fusion protein was detected in all samples. However, this fusion protein was not detectable in all 40 patients with t(8;21)-negative AMLs. The biological significance of AML1-ETO was examined in K562 cells, which stably overexpress AML1-ETO. We found that AML1-ETO blocked the erythroid differentiation of K562 cells induced by low doses of Ara-C. Thus, t(8;21)-positive AMLs appear to overexpress the AML1-ETO fusion protein, which may be responsible for differentiation block and leukemogenesis in AML.

Stable overexpression of PML alters regulation of cell cycle progression in HeLa cells.

Our previous studies demonstrated that PML is a growth suppressor that suppresses oncogenic transformation of NIH/3T3 cells and rat embryo fibroblasts. PML is a nuclear matrix-associated phosphoprotein whose expression is regulated during the cell cycle. Disruption of PML function by t(15;17) in acute promyelocytic leukemia (APL) plays a critical role in leukemogenesis. To further study the role of PML in the control of cell growth, we have stably overexpressed PML protein in the HeLa cell line. This overexpression of PML significantly reduced the growth rate of HeLa cells and suppressed anchorage-independent growth in soft agar. We consequently investigated several parameters correlated with cell growth and cell cycle progression. We found that, in comparison with the parental HeLa cells, HeLa/PML stable clones showed proportionally more cells in G1 phase, fewer cells in S phase and about the same number in G2/M phase. The HeLa/PML clones showed a significantly longer doubling time as a result of a lengthening of the G1 phase. No effect on apoptosis was found in HeLa cells overexpressing PML. This observation indicates that PML suppresses cell growth by increasing cell cycle duration as a result of G1 elongation. To further understand the mechanism of the effect of PML on HeLa cells, expression of cell cycle-related proteins in HeLa/PML and parental HeLa cells was analyzed. We found that Rb phosphorylation was significantly reduced in PML stable clones. Expression of cyclin E, Cdk2 and p27 proteins was also significantly reduced. These studies indicate that PML affects cell cycle progression by mediating expression of several key proteins that normally control cell cycle progression. These results further extend our current understanding of PML function in human cells and its important role in cell cycle regulation.

Cell-cycle regulation of DNA damage-induced expression of the suppressor gene PML.

The promyelocytic leukemia (PML) gene, which encodes a growth- and transformation-suppressor, has been identified at the non-random chromosomal translocation break point t(15; 17)(q22; q12) of acute promyelocytic leukemia. To elucidate if PML may play a role in cellular response to DNA damage, PML expression was analyzed by immunofluorescence staining in HeLa cells treated with ionizing radiation (IR) and cisplatin. Our studies demonstrated IR at 20Gy, and cisplatin at 6 micrograms/ml caused more than 5-10 fold increases in PML protein expression in the PML Oncogenic Domain (POD) by immunofluorescent staining. Northern blotting showed that there was no gross increase in mRNA levels indicating that the induction is a post-transcriptional event. Flow cytometry showed that HeLa cells treated with IR were progressively arrested in G1, which correlates with the optimal expression of PML in the cell cycle. To determine if PML expression was under the control of the tumor suppressor p53, which is known to arrest cells in G1, HeLa cells were transfected with the wild-type p53 gene. PML expression in p53 transduced cells were 5-10 fold higher than the control, indicating that the enhanced expression of PML is apparently dependent on the p53 pathway. These data also indicate that PML may play an important role in cellular response to DNA damage such as DNA repair or apoptosis during G1 arrest.

Adenovirus-mediated expression of PML suppresses growth and tumorigenicity of prostate cancer cells.

Our previous studies demonstrated that the promyelocytic leukemia gene, PML, encodes a growth and transformation suppressor. Overexpression of PML inhibits cancer cell growth in vitro and in vivo. In this study, we further explored the possibility of applying PML as a potential agent for developing prostate cancer gene therapy using an adenovirus delivery system. We have constructed and produced the recombinant PML-adenovirus, Ad-PML, in which the full-length PML cDNA is driven by the strong cytomegalovirus promoter. In LNCaP, DU145, and PC-3 prostate cancer cell lines, an infection efficiency of 90% can be achieved at a concentration of 2, 10, and 100 multiplicity of infection (MOI), respectively. Western blotting and immunofluorescence staining demonstrated that the AD-PML-infected cells expressed a high level of PML protein. The protein expression peaked at days 3-4 postinfection, and a detectable level of PML was found at day 18 after viral infection. To test the effect of Ad-PML on the growth of prostate cancer cells, the DU145 and LNCaP cells were infected with 10 and 2 MOI of Ad-PML. We found that the growth rate of the Ad-PML-infected DU145 and LNCaP cells were significantly inhibited. A tumorigenicity test in nude mice showed that the Ad-PML-treated DU145 cells failed to form tumors. Most importantly, direct injection of Ad-PML into DU145-induced tumors was able to repress tumor growth in nude mice by 64%. Taken together, these data indicate that PML is a tumor growth suppressor in prostate cancer and that Ad-PML may be a potential candidate for human prostate cancer therapy.

The biologic function of PML and its role in acute promyelocytic leukemia.

Patients with acute promyelocytic leukemia (APL) are characterized by the presence of a t(15;17) chromosomal translocation. The fusion protein PML-RAR alpha encoded from the breakpoint can form a heterodimer and acts as a dominant negative inhibitor against the normal function of PML. Recently we demonstrated that PML is a growth suppressor and transcription suppressor expressed in all cell lines tested. We also found that PML suppresses the clonogenicity and tumorigenicity of APL-derived NB4 cells, as well as the transformation of rat embryo fibroblasts by cooperative oncogenes and NIH/3T3 by neu. Overexpression of PML in human tumor cell lines induces a remarkable reduction in growth rate in vitro and in vivo. More recently, we have shown that PML is a phosphoprotein associated with the nuclear matrix and that its expression is cell cycle related. PML expression is altered during human oncogenesis, implying that PML may be an anti-oncogene involved not only in APL but also in other oncogenic events. Mutation analysis of the functional domains of PML demonstrated that its ability to form PML nuclear bodies or PODs (PML oncogenic domains) is essential for suppressing growth and transformation. In light of the above studies it appears that disruption of the normal function of PML plays a critical role in the pathogenesis of APL.

PML suppresses oncogenic transformation of NIH/3T3 cells by activated neu.

The chromosomal translocation t(15;17)(q22;q12) is a consistent feature of acute promyelocytic leukemia (APL) that results in the disruption of genes for the zinc finger transcription factor PML and the retinoic acid receptor alpha (RAR alpha). We have previously shown that PML is a growth suppressor and is able to suppress transformation of NIH/3T3 by activated neu oncogene. In the study presented here, the full-length PML cDNA was transfected into B104-1-1 cells (NIH/3T3 cells transformed by the activated neu oncogene) by retrovirally mediated gene transfer. We found that expression of PML could reverse phenotypes of B104-1-1 including morphology, contact-limiting properties, and growth rate in both transient-expression and stable transfectants. We also demonstrated that PML is able to suppress clonogenicity of B104-1-1 in soft agar assay and tumorigenicity in nude mice. These results strongly support our previous finding that PML is a transformation or growth suppressor. Our results further demonstrate that expression of PML in B104-1-1 cells has little effect on cell cycle distribution. Western blot analysis demonstrated that suppression of neu expression in B104-1-1 by PML was insignificant in the transient transfection experiment but significant in the PML stable transfectants. This study suggests that PML may suppress neu expression and block signaling events associated with activated neu. This study supports our hypothesis that disruption of the normal function of PML, a growth or transformation suppressor, is a critical event in APL leukomogenesis.

The PML gene encodes a phosphoprotein associated with the nuclear matrix.

The t(15;17)(q22;q12) translocation is the cytogenetic hallmark of acute promyelocytic leukemia (APL). The PML and retinoic acid receptor-alpha (RAR alpha) transcription factor genes are involved at translocation breakpoint. To elucidate the biologic function of PML, antipeptide antibody against PML protein was raised in rabbits. This antibody was able to detect a 90-kD PML protein and a 110-kD PML-RAR alpha fusion protein by Western blotting and a nuclear speckled pattern in all cell lines by immunofluorescent staining. In K562 and NIH/3T3 cells transfected with a PML expression plasmid, we found PML to be associated with the nuclear matrix. Our results also showed that PML is a phosphorprotein. A weak signal was detected in a Western blot containing the immunoprecipitated PML protein using the phosphotyrosine-specific monoclonal antibody. Therefore, at least one of the sites was phosphorylated by a tyrosine kinase. From our analysis of the phosphoamino acids of the PML protein by complete hydrolysis and thin-layer chromatography, we concluded that both tyrosine and serine residues of PML are phosphorylated. To investigate whether expression of the PML protein is cell-cycle related, HeLa cells synchronized at various phases of the cell cycle were analyzed by immunofluorescence staining and confocal microscopy for PML expression. We found that PML was expressed at a lower level in S, G2, and M phases and at a significantly higher level in G1 phase. Our study showed that PML has many similar properties compared with the tumor suppressor, eg, Rb. These findings further support the important role of PML in APL pathogenesis.

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

Regulation of the expression of annexin VIII in acute promyelocytic leukemia.

Annexin VIII is a calcium-dependent phospholipid-binding protein previously identified as a blood anticoagulant based on in vitro studies. However, the physiologic function of annexin VIII remains unknown. In acute promyelocytic leukemia (APL) the annexin VIII gene is highly expressed, but its expression is undetectable in the blasts of other acute leukemias. In the present investigation, we showed using the APL-derived NB4 cell line that expression of the annexin VIII gene is regulated at the transcription level during induced differentiation by all-trans retinoic acid (ATRA). The half-life of the annexin VIII mRNA is about 5 to 6 hours, as determined by using actinomycin D as a transcription inhibitor. Analysis of the expression of annexin VIII protein in NB4 cells and in APL samples showed a consistent expression of a predominant 36-kD protein and a weak 72-kD protein. After ATRA-induced differentiation of NB4 cells, the annexin VIII protein level reduced gradually, but a detectable level persisted even after 4 days of induction. Because annexin VIII mRNA becomes undetectable after 48 hours of ATRA induction, this result indicates that annexin VIII is a relatively stable protein. A multiple tissue Northern blot analysis was performed, and we found that annexin VIII is normally expressed in the placenta and the lung. Cellular localization of the annexin VIII protein was determined by immunofluorescence staining and subcellular fractionation. These results indicated that annexin VIII is predominantly localized to the plasma membrane. The annexin VIII is neither an extracellular protein nor associated with the cell surface suggesting that it does not play a role in blood coagulation in vivo. The plasma membrane localization and its property as a phospholipase inhibitor suggests that annexin VIII may have a role in the signal transduction pathway in the APL cells.