Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs.

BACKGROUND: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. METHODOLOGY/PRINCIPAL FINDING: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. CONCLUSION/SIGNIFICANCE: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

TGF-beta repressors SnoN and Ski are implicated in human colorectal carcinogenesis.

BACKGROUND: The TGF-beta signaling repressors SnoN and Ski have been critically implicated in human cancer. METHODS: To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR. RESULTS: SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with beta-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels. CONCLUSION: Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease.

Cdt1 and Geminin in cancer: markers or triggers of malignant transformation?

Cdt1 and its inhibitor Geminin are important regulators of replication licensing. In normal cells, a critical balance between these two proteins ensures that firing of each origin along the genome will take place only once per cell cycle. Cdt1 overexpression in cell lines and animals leads to aberrant replication, activates DNA damage checkpoints and predisposes for malignant transformation. Geminin inactivation mimics the effects of Cdt1 overexpression in cells and generates mitotic defects and abnormal chromosome segregation. Aberrant expression of Cdt1 and Geminin is thus linked to DNA replication defects, aneuploidy and genomic instability. These traits are considered integral to precancerous states and essential elements for malignant transformation. Moreover, Cdt1 and Geminin expression is deregulated in human tumor specimens and Cdt1 and Geminin may represent novel markers useful for cancer diagnosis and prognosis.

Arkadia enhances Nodal/TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover.

Regulation of transforming growth factor-beta (TGF-beta) signaling is critical in vertebrate development, as several members of the TGF-beta family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-beta pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous null (-/-) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia-/- cells, like Smad2-/- cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals.

Reduced expression levels of the senescence biomarker clusterin/apolipoprotein j in lymphocytes from healthy centenarians.

Clusterin/apolipoprotein J (CLU) is a conserved, ubiquitously expressed secreted glycoprotein that has been implicated in several physiological processes and was found to accumulate in many severe physiological disturbances. We have previously shown that the CLU gene and protein are upregulated during replicative senescence, stress-induced premature senescence, in vivo aging, and in several age-related diseases. In this study we have examined the CLU gene relationship to human longevity. We recruited and further analyzed 96 blood samples from Italian and Greek healthy donors of different ages, including 49 centenarians. We found that although the CLU gene expression levels increase during aging, in the centenarians' samples CLU levels were lower than those found in old donors. We then investigated the possible existence of a genetic polymorphism related to longevity at the CLU structural locus. A neutral noncoding sequence variant was detected 35 nucleotides upstream from exon 6, which does not correlate, however, with the age of the donor. We conclude that CLU gene accumulation during in vivo aging does not directly relate to chronological age, but rather indicates increased levels of organismal stress due to a progressive failure of homeostasis and/or to prolonged exposure to a stressful environment.

A novel mitochondrial DNA-like sequence insertion polymorphism in Intron I of the FOXO1A gene.

The human forkhead box O1A (FOXO1A) gene belongs to the human forkhead gene family and acts downstream of the human insulin signalling pathway. In this study, polymorphisms of the Intron I of FOXO1A gene were studied in Italian healthy people and insulin resistant subjects. No significant association between the germ-line variability in the Intron I of FOXO1A and insulin resistance was observed. Interestingly, during the study, a new 39-bp sequence insertion polymorphism in Intron I of FOXO1A gene was described. The polymorphism was found to co-segregate in a co-dominant Mendelian fashion and to be present in an ethnically distinct population (Greeks). A BLAST search showed that the sequence shares 100% identity with a mtDNA (mitochondrial DNA) sequence coding for the ATP synthase 8 (ATPase8) and ATP synthase 6 (ATPase6) genes. Hence, FOXO1A Intron I is a polymorphic nuclear region involved in the exchange of DNA material between mitochondrial and genomic DNA, which is a well-established mechanism of evolutionary change in eukaryotes.

Correlation of MDR-1, nm23-H1 and H Sema E gene expression with histopathological findings and clinical outcome in ovarian and breast cancer patients.

The development of a molecular screening method for cancer patients is of great importance, since it would contribute to the selection of the most effective chemotherapy regimen for each patient. In the present study we applied such a method, semi-quantative RT-PCR analysis, and we examined the expression of the multidrug resistance gene MDR-1, the metastasis suppressor gene nm23-H1 and the non-MDR drug resistant gene H Sema E in 53 ovarian and breast cancer specimens. Moreover, we have correlated the expression profile of these genes with the histopathological findings and clinical outcome of the examined patients. The majority of specimens were found to be positive for MDR-1 and H Sema E gene expression, while nm23-H1 was detected in less than 50% of the patients. Correlation and statistical analysis of the molecular data with clinicopathological features showed that nm23-H1 could serve as a good prognostic factor for ovarian cancer patients. In breast cancer patients, nm23-H1 expression was associated with a 6.1 higher death risk. Ovarian cancer patients who express nm23-H1, but not MDR-1 and H Sema E, tend to have longer survival than patients with any other gene combination. Finally, breast cancer patients with advanced disease showed a better response when they were negative for all the three genes studied. In conclusion this work proposes that the combined study of the expression of different genes may be a useful approach for evaluating patients' response to therapy.

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.