Smoking negatively impacts renal cell carcinoma overall and cancer-specific survival

Tobacco use is a leading cause of premature death, yet few studies have investigated the effect of tobacco exposure on the outcome of patients with renal cell carcinoma (RCC). The authors of this report retrospectively studied the impact of smoking on clinicopathologic factors, survival outcomes, and p53 expression status in a large cohort of patients with RCC.

Urothelial neoplasms of the urinary bladder occurring in young adult and pediatric patients: a comprehensive review of literature with implications for patient management

Bladder urothelial carcinoma is typically a disease of older individuals and rarely occurs below the age of 40 years. There is debate and uncertainty in the literature regarding the clinicopathologic characteristics of bladder urothelial neoplasms in younger patients compared with older patients, although no consistent age criteria have been used to define "younger" age group categories. Use of the World Health Organization 2004/International Society of Urological Pathology 1998 grading nomenclature and recent molecular studies highlight certain unique features of bladder urothelial neoplasms in young patients, particularly in patients below 20 years of age. In this meta-analysis and review, the clinical, pathologic, and molecular features and risk factors of bladder urothelial neoplasms in patients 40 years or less are presented and analyzed according to decades of presentation. Similar to older patients, bladder urothelial neoplasms in patients 40 years or younger occur more common in male patients, present mainly with gross painless hematuria, and are more commonly located at bladder trigone/ureteral orifices, but in contrast have a greater chance for unifocality. Delay in diagnosis of bladder urothelial neoplasms seems not to be uncommon in younger patients probably because of its relative rarity and the predominance of benign causes of hematuria in this age group causing hesitancy for an aggressive work-up. Most tumors in patients younger than 40 years were low grade. The incidence of low-grade tumors was the lowest in the first 2 decades of life, with incremental increase of the percentage of high-grade tumors with increasing age decades. Classification according to the World Health Organization 2004/International Society of Urological Pathology grading system identified papillary urothelial neoplasms of low malignant potential to be relatively frequent among bladder tumors of young patients particularly in the teenage years. Similar to grade, there was marked predominance of low stage tumors in the first 2 decades of life with gradual inclusion of few higher stage and metastatic tumors in the 2 older decades. Bladder urothelial neoplasms occurring in patients <20 years of age lack or have a much lower incidence of aberrations in chromosome 9, FGFR3, p53, and microsatellite instability and have fewer epigenetic alterations. Tumor recurrence and deaths were infrequent in the first 2 decades and increased gradually in each successive decade, likely influenced by the increased proportion of higher grade and higher stage tumors. Our review of the literature shows that urothelial neoplasms of the bladder occurring in young patients exhibit unique pathologic and molecular features that translate to its more indolent behavior; this distinction is most pronounced in patients <20 years. Our overall inferences have potential implications for choosing appropriate noninvasive diagnostic and surveillance modalities, whenever feasible, and for selecting suitable treatment strategies that factor in quality of life issues vital to younger patients.

Inhibition of damage-regulated autophagy modulator-1 (DRAM-1) impairs neutrophil differentiation of NB4 APL cells

The damage-regulator autophagy modulator 1 (DRAM-1) is a lysosomal protein that positively regulates autophagy in a p53-dependent manner. We aimed at analyzing the role of DRAM-1 in granulocytic differentiation of APL cells. We observed a significant increase of DRAM-1 expression during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 APL cells but not in ATRA-resistant NB4-R2 cells. Next, knocking down DRAM-1 in NB4 APL cells was sufficient to impair neutrophil differentiation. Given that DRAM-1 is a transcriptional target of p53, we tested if DRAM-1 is regulated by the p53 relative p73. Indeed, inhibiting p73 prevented neutrophil differentiation and DRAM-1 induction of NB4 cells. In conclusion, we show for the first time that p73-regulated DRAM-1 is functionally involved in neutrophil differentiation of APL cells.

Reduced nuclear translocation of nuclear factor (NF)-kappaB p65 in the footpad epidermis of dogs infected with distemper virus

Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.

Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMkappa responsible for platelet agglutination in plasma and inducing signal transduction

Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, is known to bind specifically to platelet glycoprotein (GP)Ib. We now show that, in addition, it agglutinates platelets in plasma and induces platelet signal transduction. The agglutination is caused by binding to a specific protein in plasma. The protein was isolated from plasma and shown to cause platelet agglutination when added to washed platelets in the presence of echicetin. It was identified as immunoglobulin Mkappa (IgMkappa) by peptide sequencing and dot blotting with specific heavy and light chain anti-immunoglobulin reagents. Platelet agglutination by clustering echicetin with IgMkappa induced P-selectin expression and activation of GPIIb/IIIa as well as tyrosine phosphorylation of several signal transduction molecules, including p53/56(LYN), p64, p72(SYK), p70 to p90, and p120. However, neither ethylenediaminetetraacetic acid nor specific inhibition of GPIIb/IIIa affected platelet agglutination or activation by echicetin. Platelet agglutination and induction of signal transduction could also be produced by cross-linking biotinylated echicetin with avidin. These data indicate that clustering of GPIb alone is sufficient to activate platelets. In vivo, echicetin probably activates platelets rather than inhibits platelet activation, as previously proposed, accounting for the observed induction of thrombocytopenia.

Tumour suppressors in liver carcinogenesis

The circuitous cell signalling pathways of hepatocytes comprise several factors that operate to downgrade or even interrupt the transmission of a given signal. These down-regulating influences are essential to keep cell proliferation and cell survival in check and if impaired, can alter a delicate balance in favour of cell proliferation. Each signalling pathway that has been implicated in carcinogenesis is influenced by both oncogenic factors that promote tumour growth when activated as well as tumour suppressor proteins that have to be impaired to favour tumour growth. This summary of the Tumour Suppressors in Liver Carcinogenesis Symposium held at the 2007 EASL Annual Meeting discusses four pathways with pre-eminent tumour suppressor activity, each involved in hepatocarcinogenesis: p53, mTOR, beta-catenin and hedgehog.

Clinical significance of cell cycle- and apoptosis-related markers in biliary tract cancer: a tissue microarray-based approach revealing a distinctive immunophenotype for intrahepatic and extrahepatic cholangiocarcinomas

Cholangiocarcinoma is the second most common malignant tumor of the liver. We analyzed, immunohistochemically, the significance of cell cycle- and apoptosis-related markers in 128 cholangiocarcinomas (42 intrahepatic, 70 extrahepatic, and 16 gallbladder carcinomas) combined in a tissue microarray. Follow-up was available for 57 patients (44.5%). In comparison with normal tissue (29 specimens), cholangiocarcinomas expressed significantly more frequently p53, bcl-2, bax, and COX-2 (P.05 <). Intrahepatic tumors were significantly more frequently bcl-2+ and p16+, whereas extrahepatic tumors were more often p53+ (P < .05). Loss of p16 expression was associated with reduced survival of patients. Our data show that p53, bcl-2, bax, and COX-2 have an important role in the pathogenesis of cholangiocarcinomas. The differential expression of p16, bcl-2, and p53 between intrahepatic and extrahepatic tumors demonstrates that there are location-related differences in the phenotype and the genetic profiles of these tumors. Moreover, p16 was identified as an important prognostic marker in cholangiocarcinomas.

Coupling of mutated Met variants to DNA repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH(2)-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Regulation of phosphoinositide 3-kinase expression in health and disease

Both the biology and the therapeutic potential of the phosphoinositide 3-kinase (PI3K) signalling axis have been the subject of intense investigation; however, little is known about the regulation of PI3K expression. Emerging evidence indicates that PI3K levels change in response to cellular stimulation with insulin and nuclear receptor ligands, and during various physiological and pathological processes including differentiation, regeneration, hypertension and cancer. Recently identified mechanisms that control PI3K production include increased gene copy number in cancer, and transcriptional regulation of the p110alpha PI3K gene by FOXO3a, NF-kappaB and p53, and of the PI3K regulatory subunits by STAT3, EBNA-2 and SREBP. In most instances, however, the impact of alterations in PI3K expression on PI3K signalling and disease remains to be established.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.

Tumor suppressor genes in myeloid differentiation and leukemogenesis

Tumor suppressor genes, such as p53, RB, the INK4-ARF family and PML, suppress malignant transformation by regulating cell cycle progression, ensuring the fidelity of DNA replication and chromosomal segregation, or by inducing apoptosis in response to potentially deleterious events. In myeloid leukemia, hematopoietic differentiation resulting from highly coordinated, stage-wise expression of myeloid transcription and soluble signaling factors is disrupted leading to a block in terminal differentiation and uncontrolled proliferation. This virtually always involves functional inactivation or genetic disruption of one or several tumor suppressor genes in order to circumvent their checkpoint control and apoptosis-inducing functions. Hence, reactivation of tumor suppressor gene function has therapeutic potential and can possibly enhance conventional cytotoxic chemotherapy. In this review, we focus on the role of different tumor suppressor genes in myeloid differentiation and leukemogenesis, and discuss implications for therapy.

Clinical, histologic, and immunohistochemical analyses of feline squamous cell carcinoma in situ

Actinic keratosis (AK) and Bowenoid in situ carcinoma (BISC) are two distinct forms of in situ squamous cell carcinoma in felines. They usually occur on different locations and present with specific clinical and histologic features. However, in some cases, these diseases cannot be distinguished either clinically or histopathologically. The aim of the present study was to determine the accuracy of diagnosis based on clinical or histologic criteria alone, and whether immunohistochemistry for papillomavirus or p53 can improve the accuracy of diagnosis. A series of in situ squamous cell carcinoma cases (n = 45) were selected according to their location and initial histologic classification and subsequently classified as AK (n = 22) or BISC (n = 23) according to the clinical criteria and were reevaluated histologically by 2 dermatopathologists. All BISC cases and most of the AK cases (n = 15) were confirmed histologically. In 7 cases clinically classified as AK, this diagnosis was not unanimously confirmed histologically because of the presence of overlapping features. P53 immunoreactivity was observed in 11/14 (79%) confirmed AK cases and in 4/22 (18%) BISC cases, while papillomavirus antigen was not detected in any confirmed AK case but was detected in 11/23 (48%) BISC cases. It was concluded that BISC can usually be reliably diagnosed histologically. The histologic diagnosis of lesions clinically suggestive of AK might sometimes be difficult. Results of immunohistochemistry for p53 and papillomavirus antigen were supportive for a role of sun exposure and papillomavirus in the pathogenesis of AK and BISC, respectively.

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8

Background: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods: A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.