Conventional cytology, visual tests and evaluation of P16(INK4A) as a biomarker in cervical intraepithelial neoplasia.

OBJECTIVES: (1) To detect cervical intraepithelial neoplasia (CIN) using Papanicolaou test (PAP test), visual tests (visual inspection after the application of acetic acid [VIA], visual inspection after the application of Lugol's iodine [VILI]), colposcopy, and biopsy. (2) To study the biomarker p16(INK4A) expression by immunostaining. MATERIALS AND METHODS: Experimental study was conducted from November 2009 to April 2011. 1500 women were screened for cancer cervix using conventional PAP test, VIA, and VILI. Sensitivity, specificity, positive, and negative predictive values of these tests were calculated individually, sequentially, and in parallel. Women having positive results underwent colposcopy and biopsy if required. p16(INK4A) expression in biopsy samples was studied using immunohistochemistry. RESULTS: All test positive cases (n = 235) underwent colposcopy. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PAP with atypical squamous cells of undetermined significance (ASCUS) as cut-off was 40%, 99.25%, 35.25%, and 99.39%; VIA was 60%, 93.06%, 8.03%, and 99.56% and VILI was 80%, 86.06%, 5.4%, and 99.76%, respectively. When PAP, VIA, and VILI were used in parallel sensitivity, specificity, PPV, and NPV improved to 100%, 85.18%, 6.38%, and 100%, respectively. Colposcopic abnormalities were detected in 83 and biopsy proven CIN in 15. p(16INK4A) expression was seen in eight of 15 CIN cases. CONCLUSIONS: (1) PAP test and visual techniques are complementary. (2) p(16INK4A) expression was seen in majority of CIN 2 lesions suggesting a higher grade lesion.

Topical hypochlorite ameliorates NF-κB-mediated skin diseases in mice.

Nuclear factor-κB (NF-κB) regulates cellular responses to inflammation and aging, and alterations in NF-κB signaling underlie the pathogenesis of multiple human diseases. Effective clinical therapeutics targeting this pathway remain unavailable. In primary human keratinocytes, we found that hypochlorite (HOCl) reversibly inhibited the expression of CCL2 and SOD2, two NF-κB-dependent genes. In cultured cells, HOCl inhibited the activity of inhibitor of NF-κB kinase (IKK), a key regulator of NF-κB activation, by oxidizing cysteine residues Cys114 and Cys115. In NF-κB reporter mice, topical HOCl reduced LPS-induced NF-κB signaling in skin. We further evaluated topical HOCl use in two mouse models of NF-κB-driven epidermal disease. For mice with acute radiation dermatitis, topical HOCl inhibited the expression of NF-κB-dependent genes, decreased disease severity, and prevented skin ulceration. In aged mice, topical HOCl attenuated age-dependent production of p16INK4a and expression of the DNA repair gene Rad50. Additionally, skin of aged HOCl-treated mice acquired enhanced epidermal thickness and proliferation, comparable to skin in juvenile animals. These data suggest that topical HOCl reduces NF-κB-mediated epidermal pathology in radiation dermatitis and skin aging through IKK modulation and motivate the exploration of HOCl use for clinical aims.

Ginsenoside Rb1, Rg1 and three extracts of traditional Chinese medicine attenuate ultraviolet B-induced G1 growth arrest in HaCaT cells and dermal fibroblasts involve down-regulating the expression of p16, p21 and p53.

OBJECTIVE: The aims of this study were to confirm whether traditional Chinese medicine ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), polygonum multiflorum (PM), ginkgo extract (GE) and lycium barbarum polysaccharide (LBP) can attenuate G1 growth arrest of HaCaT cells and dermal fibroblasts induced by 10 subcytotoxic ultraviolet B (UVB) exposures, and to explore the possible mechanism in terms of the expression of cell-cycle regulatory proteins p16, p21 and p53. METHODS: Ten subcytotoxic exposures to UVB induced G1 growth arrest of HaCaT cells and dermal fibroblasts. Cell-cycle analysis was performed using flow cytometry, and mRNA levels of p16, p21 and p53 were detected by a reverse transcription-polymerase chain reaction (RT-PCR), and protein levels were detected using Western blot analysis. RESULTS: Five types of traditional Chinese medicine attenuated UVB-induced G1 growth arrest. The mRNA and protein levels of p16, p21 and p53 in HaCaT cells and dermal fibroblasts increased after UVB irradiation, but pretreatment with five types of traditional Chinese medicine decreased the expression of p16, p21 and p53. CONCLUSIONS: These results indicated that five types of traditional Chinese medicine can attenuate G1 growth arrest of HaCaT cells and dermal fibroblasts induced by UVB exposures, which was caused by down-regulating the expression of cell-cycle regulatory proteins p16, p21 and p53.

Expression of cyclin D1 and p16 in psoriasis before and after phototherapy.

BACKGROUND: Psoriasis vulgaris (PV) is characterized by keratinocyte hyperproliferation. Altered expression of cell-cycle regulatory genes involved in the cyclin D1 / p16 INK4-pRb pathway may contribute to this epidermal hyperproliferation. AIM: To assess the expression of cyclin D1 and p16 in psoriasis, and to evaluate the effect of phototherapy on their expression. METHODS: The study population comprised 25 patients with PV and 10 healthy controls. Patients were treated with 24 sessions of either narrowband ultraviolet (UV) B or psoralen UVA. Skin biopsies were taken from the affected skin of each patient before and after treatment, and from the healthy controls, to examine cyclin D1 and p16 expression. RESULTS: Before phototherapy, the mean value of cyclin D1 concentration in patients was significantly greater than that in controls and the mean value of p16 concentration in patients was significantly lower than that in controls. Following treatment, we detected a significant decrease in cyclin D1 and a significant increase in p16. CONCLUSION: Cyclin D1 upregulation and p16 downregulation may play a role in the pathogenesis of psoriasis. Normalization of the levels of both parameters may be a mechanism by which phototherapy induces remission in psoriasis.

Adding the p16(INK4a) marker to the traditional 3-marker (ER/Vim/CEA) panel engenders no supplemental benefit in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

Endocervical adenocarcinomas (ECAs) and endometrial adenocarcinomas (EMAs) are malignancies that affect the uterus; however, their biologic behaviors are quite different. This distinction has clinical significance, because the appropriate therapy may depend on the site of tumor origin. In this study, we not only compare the individual expression status of 4 immunomarkers [estrogen receptor (ER), vimentin (Vim), carcinoembryonic antigen (CEA), and p16], but also evaluate whether p16 adds value to the ER/Vim/CEA panel characteristics and performance in distinguishing between primary ECA and EMA. A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 38 hysterectomy specimens, including 14 ECAs and 24 EMAs. Tissue microarray sections were immunostained with 4 antibodies, by the avidin-biotin complex method for antigen visualization. The staining intensity and area extent of the immunohistochemical reactions were evaluated using the semiquantitative scoring system. The 3 markers (ER, Vim, CEA) and their respective panel expressions showed statistically significant (P<0.05) frequency differences in ECA and EMA tumors. The p16 marker also revealed a significant frequency difference (P<0.05) between ECA and EMA, but did not demonstrate any supplementary benefit to the traditional 3-marker panel. In conclusion, when histomorphologic and clinical doubt exist as to the primary site of origin, we suggest that the conventional 3-marker (ER/Vim/CEA) panel is appropriate. Ancillary p16-marker testing does not add value to the 3-marker panel in distinguishing between primary ECA and EMA.

Beta-hydroxyisovalerylshikonin has a profound anti-growth activity in human endometrial and ovarian cancer cells.

OBJECTIVES: Beta-hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in some lines of human tumor cells. We investigated the effect of beta-HIVS on three endometrial cancer cell lines, two ovarian cancer cell lines, and normal human endometrial epithelial cells. METHODS: Endometrial and ovarian cancer cells were treated with various concentrations of beta-HIVS, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. RESULTS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of beta-HIVS, although normal endometrial epithelial cells were viable after treatment with the same doses of beta-HIVS that induced growth inhibition in endometrial and ovarian cancer cells. Cell-cycle analysis indicated that their exposure to beta-HIVS decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. CONCLUSIONS: These results suggest that the anticancer activity of beta-HIVS may occur with higher sensitivity of cancer cells compared with normal healthy cells, when using low concentration, rising hopes that beta-HIVS may become a useful adjuvant therapy for endometrial and ovarian cancers.

[Effects of recipes for replenishing qi and activating blood on senescence related gene expressions in the liver of aging rats].

OBJECTIVE: To observe the effects of recipes for replenishing qi and activating blood on p16, p21, proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E gene expressions in the liver of aging rats. METHODS: A recipe for replenishing qi and a recipe for activating blood were administered to aging rats respectively, and the effects of the above recipes on the expressions of senescence related genes (p16, p21, PCNA, cyclin D1 and cyclin E) were examined by RT-PCR and Western blotting methods. RESULTS: The expressions of p16, p21 and cyclin D1 mRNAs and proteins in the liver of the untreated aging rats were up-regulated, while the expressions of PCNA and cyclin E mRNAs and proteins decreased. As compared with the untreated aging rats, both recipes could down-regulate the expressions of cyclin D1 mRNA and protein and up-regulate the expressions of cyclin E mRNA and protein, but had no obvious effects on the expressions of mRNAs and proteins of p16, p21 and PCNA. CONCLUSION: Recipes for replenishing qi and activating blood can improve the liver cell proliferation of aging rats via down-regulating the expressions of cyclin D1 mRNA and protein and up-regulating the expressions of cyclin E mRNA and protein.

Immunohistochemical evaluation of thymidylate synthase (TS) and p16INK4a in advanced colorectal cancer: implication of TS expression in 5-FU-based adjuvant chemotherapy.

BACKGROUND: Our previous analyses on the expression of thymidylate synthase (TS) and p16(INK4a) in colorectal cancer patients administered 5-fluorouracil (5-FU) pre-operatively demonstrated that a high level of TS expression was a predictor of 5-FU resistance, and that the combination of a low level of TS expression and induction of p16(INK4a) after chemotherapy implicated chemosensitivity. The present study aimed to assess the relationship between the biological behavior of advanced colorectal cancer treated post-operatively by 5-FU-based chemotherapy and the expression of TS and p16(INK4a) in primary tumors. METHODS: Formalin-fixed, paraffin-embedded specimens from 132 colorectal cancers (Dukes' B, 36 cases; Dukes' C, 60 cases; and Dukes' D, 36 cases) treated by 5-FU post-operatively were immunostained for TS and p16(INK4a). Antigenicities were suitably retrieved. RESULTS: Primary tumors expressing high levels of TS in the Dukes' C group showed a significantly shorter recurrence-free interval (RFI) (P = 0.0002). The overall survival (OS) was shorter in high TS expressors than in low TS expressors (P = 0.001). A high level of TS expression also correlated with advanced Dukes' staging and the severity of nodal metastasis (Dukes' B versus Dukes' D, P = 0.001; Dukes' C versus Dukes' D, P = 0.008; N0 versus N2, P = 0.002; N1 versus N2, P = 0.03). p16(INK4a) expression was not correlated with the prognosis or clinicopathological features. CONCLUSIONS: Appropriate immunohistochemical evaluation is essentially important. We suggest that, in the Dukes' C group, a 5-FU-based regimen can be chosen as a first-line chemotherapy for low TS expressors. TS-high cancer should be treated with anti-cancer agents acting through different mechanisms. Further research should be conducted on applying TS immunostaining to the treatment strategy.

5-Fluorouracil or gemcitabine combined with adenoviral-mediated reintroduction of p16INK4A greatly enhanced cytotoxicity in Panc-1 pancreatic adenocarcinoma cells.

BACKGROUND: Pancreatic cancer is one of the most lethal of all the common gastrointestinal malignancies. Although surgery offers the best chance for survival, it is not appropriate for all cases. The only adjuvant treatment to show promise is chemotherapy. Hence new treatments are urgently sought. We previously reported that adenoviral (Ad)-mediated delivery of p53 (Adp53) and p16(INK4A) (Adp16) significantly inhibited the growth of pancreatic cancer cell lines and established subcutaneous pancreatic tumours in nude mice (Ghaneh P, et al. Adenovirus mediated transfer of p53 and p16INK4A results in pancreatic cancer regression in vitro and in vivo. Gene Ther 2001; 8: 199-208). In this study we examine whether combining Ad-mediated delivery of p53 or p16(INK4A) with clinically relevant chemotherapeutic drugs has therapeutic potential for pancreatic cancer. METHODS AND RESULTS: Four pancreatic adenocarcinoma cell lines were evaluated for their sensitivity to 5-fluorouracil (5-FU) and gemcitabine and two of these, Suit-2 and Panc-1, were chosen for combination experiments because they showed moderate and poor sensitivity, respectively, to 5-FU and gemcitabine. We found no evidence for enhanced cytotoxicity when either cell line was transduced with Adp53 before or after incubation with chemotherapeutic drugs. In contrast, incubation of Panc-1 cells with either 5-FU or gemcitabine followed by Ad-mediated overexpression of p16(INK4A) resulted in a substantial reduction in cell viability under conditions where the drugs alone had minimal cytotoxicity. Incubation of Suit-2 cells with 5-FU followed by Ad-mediated overexpression of p16(INK4A) also resulted in a significant reduction in cell viability. This, however, was observed only with higher concentrations of 5-FU and viral vector. Cell cycle analysis of Panc-1 cells showed that the combination of cytotoxic drugs and Adp16 resulted in an increase in the sub-G1 population suggesting an increase in apoptosis. Dual labelling of these cells with annexin V and propidium iodide (PI) confirmed that the combination of 5-FU and Adp16 resulted in a significant increase in early apoptotic cells (annexin V positive and PI negative) compared with controls. Moreover, overexpression of p16(INK4A) was associated with a reduction in pRb levels in these cells-high levels of pRb have been proposed to contribute to chemoresistance in pancreatic cancer cells. CONCLUSIONS: We have shown that the currently used chemotherapeutic drugs for pancreatic adenocarcinoma combined with restoration of p16(INK4A) expression hold promise for the adjuvant treatment of this disease. Importantly, the combination facilitated the use of chemotherapeutic drugs at lower concentrations than would otherwise be effective.

[Experimental study on effect of epimedium flavonoids in protecting telomere length of senescence cells HU].

OBJECTIVE: To investigate the mechanism of senescence delay of human diploid fibroblast (2BS) and protecting telomere length by epimedium flavonoids (EF). METHODS: The drug sera of EF were used to treat the 2BS. The population doublings of 2BS cells were observed, the mRNA expression of p16 gene were determined by fluorescence real-time quantitative RT-PCR, the telomerase activation of 2BS cells were determined by TRAP-Hyb, the total retinoblastoma (Rb) and phosphorated Rb protein content were detected by ELISA, the telomere length of 2BS cells were determined by telomere restriction fragment (TRF) Southern blot assay. RESULTS: EF could significantly extend the population doublings of 2BS cells, the expression of p16 mRNA was decreased and the content of phosphorated Rb protein were increased by EF. The telomere lengthening of 2BS cells were improved by EF, but the telomerase was not activated. CONCLUSION: In senescence human fibroblasts 2BS cells, p16 gene mRNA expression increased, content of phosphorated Rb protein decreased and the telomere length of 2BS shortened, EF might delay the aging of cells through inhibiting the p16 gene expression, promoting the production of phosphorated Rb protein and to protect the length of telomere, but not activating the telomerase.

[Comparative study on the regulatory effects on senescence related cell cycle gene expression by TCM principles of tonifying shen, invigorating pi benefiting qi, and activating blood circulation].

OBJECTIVE: To compare the effects of TCM therapeutic principles of tonifying Shen (TS), benefiting Qi (BQ), invigorating Pi (IP) and activating blood circulation (ABC) herbs in regulating the gene expression in senescence related cell cycle. METHODS: Drug sera containing TCM herbs of the above-mentioned principles were used to treat the aged human diploid fibroblast cell line 2BS. The effect of TCM on the senescence related cell cycle and its related gene expression (P16INK4, Cyclin D1 and PCNA) were examined by means of cell proliferative doublings, flow cytometry, RT-PCR and Western blot analysis. RESULTS: TCM herbs of TS and BQ could improve the cell cycle, down-regulate the P16 and Cyclin D1 mRNA/protein expression, up-regulate PCNA mRNA/protein expression, while TCM herbs of IP and ABC showed insignificant effect on these indexes. CONCLUSION: TCM herbs of TS and BQ have effect in improving cell cycle, it may be achieved through promoting the P16 pathway of gene expression.

BCL-6 expression predicts improved survival in patients with primary central nervous system lymphoma.

PURPOSE: The purpose of this study was to investigate the histogenetic origin of primary central nervous system lymphoma (PCNSL) with respect to stage of B-cell differentiation and to identify prognostic markers in a cohort of patients with PCNSL treated with i.v. high-dose methotrexate therapy. EXPERIMENTAL DESIGN: This study included 33 patients with PCNSL treated with high-dose i.v. methotrexate at the Massachusetts General Hospital for whom archival tumor tissue was available. All 33 patients tested negative for HIV. The lymphomas were morphologically subclassified according to the Kiel system, as modified in the WHO classification. Immunohistochemistry for the following antigens was performed: BCL-6; BCL-2; MUM1; CD10; vs38c; CD138; CD44; p16; and p53. Fluorescence in situ hybridization and multiplex PCR for CDKN2A/p16 were also performed. RESULTS: There were 17 women and 16 men enrolled, with a median age of 60 years. All tumors were diffuse large B-cell lymphomas. Of the 23 cases that could be subclassified, 22 were centroblastic, and 1 was immunoblastic. Twenty-six of 33 tumors were BCL-6+, 6 of 32 tumors were CD10+, 27 of 29 tumors were BCL-2+, 31 of 32 tumors were MUM1+, 11 of 31 tumors were CD44+, 4 of 33 tumors were vs38c+, and 0 of 32 tumors were CD138+. There were 18 of 32 (56%) complete responses and 8 of 32 (25%) partial responses to methotrexate, whereas 6 of 33 (18%) progressed during treatment. Ten patients died of disease. Expression of BCL-6 was significantly associated with longer overall survival (P = 0.002; median survival, 101 versus 14.7 months, with approximately 95% lower confidence limits of 41.7 and 8.8 months, respectively). CONCLUSIONS: In this group of 33 patients with PCNSL, expression of BCL-6 was significantly associated with longer overall survival. BCL-6 warrants further investigation as a potentially important prognostic marker in this disease.

[Regulation of Chinese medicine bailong and hexamethylene bisacetamide on cyclin dependent kinase inhibitor P16 genes in human cancer cell].

OBJECTIVE: To study the regulation of bailong, a Chinese herbal anticancer preparation and hexamethylene bisacetamide (HMBA) on cyclin dependent kinase inhibitor P16 (CKI-P16) genes in human cancer cells. METHODS: The expression of CKI-P16 in different human cancer cells treated by Bailong or HMBA under different condition, was examined using Northern hybridization, Western blotting assay, etc. RESULTS: After being treated by Bailong or HMBA, the P16 expression increased. This effect was closely related to co-regulation of cAMP-PKA and DAC-PKC signal pathway. When PKA pathway was blocked with PKA inhibitor, the P16 expression decreased, while PKC pathway was blocked, it enhanced. CONCLUSION: (1) The low P16 expression in G1 phase of cancer cell, as compared with that in S, G2 and M phases, might be an important factor responsible to the incompetence of P16 in inhibiting effectively the malignant change of cancer cell. (2) Mechanism of Bailong and HMBA on cancer cell proliferation inhibition might be correlated with the enhancement of P16 expression in G1 phase of cancer cells. (3) Regulation and expression of Bailong and HMBA on P16 showed the common character of Chinese and western medicine in regulating cancer cells. (4) This study elucidated that upstream of P16 was related to cAMP/PKC signal pathway closely.