p16INK4a Expression Levels Research Articles

Accelerated Cellular Senescence in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a group of hematopoietic stem/progenitor cell clonal disorders characterized by dysplastic morphology of at least one cell lineage and high risk of evolution to acute myeloid leukemia (AML). Thus, it could be viewed as a preneoplastic stage prior to development of overt acute myeloid leukemia (AML). A number of studies indicated that oncogene-induced senescence as a crucial cellular response at premalignant stage can protect cells from tumor formation. Senescent cells emerged in premalignant cell population but not in malignant ones. p16INK4a have been identified as a cellular senescence-associated molecular marker. In present study, we have determined and compared the expression level of p16INK4a by quantitative RT-PCR and western blot in BM mononuclear cells (BMMNCs)/CD34+ cells among 53 patients of MDS and 12 of AML as well as 11 healthy controls. A significantly upregulated expression level of p16INK4a in MDS, while an significantly lower p16INK4a expression level in AML had been detected in comparison with that in healthy controls(<0.05). The IPSS score of these MDS patients negatively correlated with the p16INK4a expression level. To further confirm the senescent status, the cells of MDS, AML patients and healthy controls were examined by histochemical assays for the activity of senescence-associated beta galactosidase (SA-β-Gal). A weak blue stain reflecting the SA-β-Gal activity could be found in less than 10% cells from healthy controls after four days' culture. On the contrary, a much stronger blue stain for SA-β-Gal had been detected in about 30–40% cells from MDS, and a little-to-no blue stain in AML cells. The semi-quantitative RT-PCR had demonstrated a similar pattern of SA-β-Gal expression among these three groups. Our preliminary results raise the question whether an accelerated cellular senescence in addition to apoptosis may contribute to the hematopoietic ineffectiveness and dysplastic morphology of cells in MDS, and if it could be of any implications for the progression of the disease.

P16INK4a Expression and Absence of Activated B-RAF Are Independent Predictors of Chemosensitivity in Melanoma Tumors

Metastatic cutaneous melanoma is highly resistant to cytotoxic drugs, and this contributes to poor prognosis. In vivo studies on the chemosensitivity of metastatic melanoma are rare and hampered by poor response rates to systemic chemotherapeutics. Patients who undergo isolated limb infusion (ILI) with cytotoxic drugs show high response rates and are, therefore, a good cohort for studying chemosensitivity in vivo. We used tumors from patients who underwent ILI to study the role of melanoma tumor-suppressor genes and oncogenes on melanoma chemosensitivity. Prospectively acquired tumors from 30 patients who subsequently underwent ILI with melphalan and actinomycin-D for metastatic melanoma were investigated for mRNA expression levels of p14ARF, p16INK4a, and MITFm. The mutation status of B-RAF, N-RAS, and PTEN were also determined. A high percentage of tumors had activating mutations in either B-RAF (15/30) or N-RAS (10/30) and only two tumors carried altered PTEN. High expression of p16INK4a and absence of an activating B-RAF mutation independently predicted response to treatment. Further, inducible expression of p16INK4a sensitized a melanoma cell line to death induced by melphalan or actinomycin-D. This study shows that high expression of p16INK4a or the absence of activated B-RAF correlates with in vivo response of melanoma to cytotoxic drugs.

The methylation pattern of p16INK4agene promoter in psoriatic epidermis and its clinical significance

Alteration of the p16INK4a gene by epigenetic changes has been described in some hyperproliferative skin diseases, but its importance in psoriasis has not yet been established. To investigate the methylation status of the p16INK4a gene in psoriatic epidermis, its clinical significance and the possible epigenetic mechanisms of psoriasis. DNA and RNA specimens were obtained from the lesional epidermis of 56 patients with plaque psoriasis. Methylation-specific polymerase chain reaction (PCR) and DNA sequencing were used to detect the density and sites of methylation in the p16INK4a promoter region. The reverse transcription-PCR technique was applied to detect the mRNA expression of p16INK4a. p16INK4a gene promoter methylation was shown in 17 of 56 (30%) patients with psoriasis. Psoriasis Area and Severity Index scores in patients showing methylation were higher than in those who did not (P<0.05). The mRNA expression level of p16INK4a in the methylated group was significantly lower than in the unmethylated group (t=2.515, P=0.015). In the methylated group, about 50% of the CpG islands were methylated in the promoter region. Overall, methylation of the p16INK4a gene promoter is found in psoriatic epidermis, which is associated with the mRNA level of p16INK4a expression and activity of the disease. These data indicate that methylation of the p16INK4a promoter may play a potential role in the pathogenesis of psoriasis.

The role of noncartilage‐specific molecules in differentiation of cartilaginous tumors

Chondroblastoma (CB) and chondromyxoid fibroma (CMF) are benign tumors of bone morphologically recapitulating cartilage differentiation. CMF can resemble high-grade central chondrosarcoma (HGCCS) because of its cellular atypia. The mechanism that drives this morphologic spectrum of cartilage differentiation is unclear. CMFs and CBs were hybridized on a complementary DNA microarray that was enriched for cartilage-specific genes. Data were analyzed by Linear Model for Microarray Analysis and were compared with previous data on osteochondromas and HGCCS. Verification was performed in an extended series. None of the 68 genes that were differentially expressed in CB versus CMF, including several extracellular matrix (ECM) and ECM-degradation genes, were related specifically to cartilage. Perlecan, versican, collagen 4A2 (Col4A2), and cell-cell adhesion genes, such as CD166, were significantly higher in CMF. Sixty genes were expressed differentially in CMF versus HGCCS. Higher expression levels of CD166, cyclin D1 (CCND1), and p16INK4A were observed in CMF. The current findings indicated that differential expression of adhesion and ECM molecules, such as CD166, versican, perlecan, and Col4A2, may interfere with cartilaginous differentiation. The decreased expression of CCND1, p16INK4A, and CD166 in HGCCS reflects impairment of cell cycle progression and of cell-cell adhesions in malignant tumors and is of use in the differential diagnosis of CMF.

Expression of p16 INK4a in relation to histopathology and viral load of ‘high-risk’ HPV types in cervical neoplastic lesions

A total of 91 cervical archival biopsy series were analysed for the presence and viral load of ‘high-risk’ types of human papillomavirus (HR-HPV), and p16 INK4a expression. The women had various degrees of CIN (cervical intraepithelial neoplasia). HPV 16 was the most prevalent type found, at 47% frequency. The frequency of HPV 16 increased with increasing immunoreactivity to p16 INK4a, from 39% to 44% at cases scored low to medium, to 65% at high reactivity. Thirty (33%) of the samples had negative p16 INK4a analysis results, but were positive for HR-HPV. There was no significant correlation between viral load and the level of p16 INK4a expression, while the grade of CIN correlated to such expressions. Thus, p16 INK4a expression analysis yielded information which is consistent with results from the histopathology and might complement the HPV analysis in a clinical prognostic procedure in order to find women at risk for cervical cancer.

Alveolar Cell Senescence in Patients with Pulmonary Emphysema

The prevalence of chronic obstructive pulmonary disease (COPD) is age-dependent, suggesting an intimate relationship between the pathogenesis of COPD and aging. In this study we investigated whether the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. Samples of lung tissue were obtained from patients with emphysema, asymptomatic smokers, and asymptomatic nonsmokers. Paraffin-embedded lung tissue sections were evaluated for cellular senescence by quantitative fluorescence in situ hybridization to assess telomere shortening, and by immunohistochemistry to assess the expression of senescence-associated cyclin-dependent kinase inhibitors. Tissue sections were also immunostained for proliferating cell nuclear antigen (PCNA), surfactant protein A, and CD31. The patients with emphysema had significantly higher percentages of type II cells positive for p16INK4a and p21CIP1/WAF1/Sdi1 than the asymptomatic smokers and nonsmokers. They had also significantly higher percentages of endothelial cells positive for p16INK4a than the asymptomatic smokers and nonsmokers, and higher percentages of endothelial cells positive for p21CIP1/WAF1/Sdi1 than the asymptomatic nonsmokers. Telomere length in alveolar type II cells and endothelial cells was significantly shorter in the patients with emphysema than in the asymptomatic nonsmokers. The level of p16INK4a expression was negatively correlated with the level of PCNA expression. The level of alveolar cell senescence was positively correlated with airflow limitation. These results suggest that the senescence of alveolar epithelial and endothelial cells is accelerated in patients with emphysema. Cellular senescence may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.

Immunohistochemical Expression of p16INK4a and bcl-2 According to HPV Type and to the Progression of Cervical Squamous Intraepithelial Lesions

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.

Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients

Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16 INK4A and p14 ARF that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14 ARF / p16 INK4A , concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16 INK4A and p14 ARF were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.

Expression of the p14ARF tumor suppressor predicts survival in acute myeloid leukemia.

Cell cycle aberrations are associated with therapy outcome in many types of cancer. We analyzed mRNA expression levels of 18 cell cycle-related genes in bone marrow samples from 78 acute myeloid leukemia (AML) patients and six controls using high-throughput quantitative RT-PCR. Samples of AML patients contained significantly increased mRNA expression levels of the mdm2 and c-myc oncogenes. Also, the average expression levels of p14ARF and p16INK4A were higher in patient samples compared to controls. Leukemic blasts and control bone marrow samples did not differ significantly in the expression levels of proliferation-associated genes such as cyclin A2 and pcna. When single genes were analyzed for prognostic significance in Kaplan-Meier and Cox regression analyses, a low p14ARF level emerged as a strong and independent predictor for poor survival (P=0.04 and 0.029). Subsequently, p14ARF mRNA levels were analyzed in a second, independent patient population (n=57). Again, low p14ARF levels were associated with a worse outcome. Finally, immunohistochemistry analysis of AML tissue arrays confirmed the widespread expression of c-myc and p14ARF in AML on the protein level. Taken together, the expression of the p53 regulators mdm2 and p14ARF are altered in AML, and low p14ARF levels indicate a poor prognosis.

Alteration of keratinocyte differentiation and senescence by the tumor promoter dioxin☆

Exposure to the environmental contaminant dioxin, elicits a variety of responses, which includes tumor promotion, embryotoxicity/teratogenesis, and carcinogenesis in both animals and humans. Many of the effects of dioxin are mediated by the aryl hydrocarbon receptor (AHR), a ligand-activated bHLH (basic helix-loop-helix)/PAS transcription factor. We initiated this study to determine whether dioxin's tumor-promoting activities may lie in its ability to alter proliferation, differentiation, and/or senescence using normal human epidermal keratinocytes (HEKs). Here, we report that dioxin appears to accelerate differentiation as measured by flow cytometry and by increased expression of the differentiation markers involucrin and filaggrin. In addition, dioxin appears to increase proliferation as indicated by an increase in NADH/NADPH production and changes in cell cycle. Finally, dioxin decreases SA (senescence associated) β-galactosidase staining, an indicator of senescence, in the differentiating keratinocytes. These changes were accompanied by decreases in the expression levels of key cell cycle regulatory proteins p53, p16 INK4a, and p14 ARF. Our findings support the idea that dioxin may exert its tumor-promoting actions, in part, by downregulating the expression levels of key tumor suppressor proteins, which may impair the cell's ability to maintain its appropriate cellular status.

Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway.

Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts.

The occurrence of DNA double strand breaks induces cell cycle arrest in mortal and immortal human cells. In normal, mortal fibroblasts this block to proliferation is permanent. It depends on the growth regulator p53 and a protein p53 induces, the cyclin dependent kinase inhibitor, p21. We show here that following DNA damage in mortal fibroblasts, the induction of p21 and p53 is to a large degree shortlived. By 8 days after a brief exposure to DNA strand breaking agents, bleomycin or actinomycin D, p53 protein is at baseline levels, while the p53 transactivation level is only slightly above its baseline. By this time the concentration of p21 protein, which goes up as high as 100-fold shortly after treatment, is down to just 2-4-fold over baseline levels. Following the drop in p21 concentration a large increase in the expression level of the tumor suppressor gene p16INK4a is observed. This scenario, where a transient increase in p21 is followed by a delayed induction of p16INK4a, also happens with the permanent arrest that occurs with cellular senescence. In fact, these cells treated with agents that cause DNA double strand breaks share a number of additional markers with senescent cells. Our findings indicate that these cells are very similar to senescent cells and that they have additional factor(s) beside p21 and p53 that maintain cell cycle arrest.

A hermetically sealed 20-GHz integrated mixer : T. Araki, M. Hirayama, N. Fujine and T. Kajimura. NEC Res. Develop. 81 (Apr. 1974)

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors.The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging.By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen–glycosaminoglycan–chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages.We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model.To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.