Ki67 Gene Expression Research Articles

A critical role of Sp1 transcription factor in regulating the human Ki-67 gene expression

Ki-67 plays a crucial role in cell proliferation as well as maintenance or regulation of cell division. The mechanism governing the Ki-67 gene expression remains unknown. Thus, we cloned the core promoter of the human Ki-67 gene and further investigated its transcriptional regulation. The putative Sp1 binding sites were confirmed by electrophoretic mobility shift assay together with an anti-Sp1 antibody-mediated supershift assay. Deletion mutagenesis and firefly luciferase reporter gene assay demonstrated the essential contribution of Sp1 on transcriptional activation of the Ki-67 gene. In this study, we first confirm that there are three Sp1 binding sites in the Ki-67 core promoter. Two Sp1 sites (one at position -159 to -145 nt and the other at position -14 to +12 nt) are mainly involved in transcriptional regulation of the Ki-67 gene. Overexpression of Sp1 can enhance the Ki-67 promoter activity. However, down-regulation of Sp1 expression using siRNA-Sp1 and mithramycin effectively inhibits the Ki-67 gene transcription. Our results suggest that Sp1 is essential for basal promoter activity of the human Ki-67 gene. Inhibition of the Ki-67 transcriptional activity through abolishment of Sp1 may provide the useful prospect for gene therapy.

Fascin is a predictor for invasiveness and recurrence of urothelial carcinoma of bladder

ObjectivesTo evaluate the expression of fascin in bladder urothelial carcinoma, and to analyze its association with clinicopathologic features and prognosis of urinary bladder urothelial carcinoma. Materials and methodsImmunohistochemistry was used to detect the expression of fascin, Ki-67, p53, CK20, and multidrug resistance gene (MDR) in 111 bladder urothelial carcinoma and 42 normal epithelial tissues. The association between fascin expression and clinicopathologic parameters and prognostic factors on tumor recurrence was analyzed by Kaplan-Meier method, log-rank test, and Cox proportional hazards model. ResultsNinety-four of 111 cases of bladder urothelial carcinoma showed positive fascin expression, while no fascin expression was detected in normal transitional epithelium. There was a significant difference in the expression of fascin in normal epithelium and bladder urothelial carcinoma (P = 0.000). Fascin expression was positively correlated with pT stage (P = 0.001) and tumor size (P = 0.011), while it had no association with age, gender, and tumor grade (P > 0.05). pT stage and the expression of fascin, Ki-67, p53, and CK20 were significantly correlated with urothelial carcinoma recurrence, and fascin expression was an independent factor predicting tumor recurrence. ConclusionsFascin expression was up-regulated in bladder urothelial carcinoma. Over-expression of fascin might play an important role in invasiveness and recurrence of bladder urothelial carcinoma. Fascin may be used as a prognostic marker and a new target for the treatment of bladder urothelial carcinoma.

Abstract B47: Potential role for RNF8 in breast cancer development

Abstract Background: Women of Mexican descent have a higher mortality and higher proportion of triple negative or basal-like breast cancers with BRCA like features and a higher population burden of BRCA mutations. Basal-like breast cancer subtype is associated with an elevated Ki67 index and extensive genomic instability. This increase in the Ki67 index indicates cell proliferation and perhaps greater cell DNA damage. In another report it was shown that knockdown of ring finger 8 (RNF8), an E3 ubiquitin-protein ligase, by siRNA inhibited focus formation at double strand break sites suggesting that RNF8 is an important regulator of the DNA repair system. We hypothesized that loss of RNF8 would be associated with more aggressive tumors such as those having high Ki67 levels. We are investigating the hypothesis that RNF8 is a tumor suppressor gene and acts as a novel regulatory protein of BRCA1 expression in human breast cancers. Methods: We tested whether 37 formalin fixed paraffin embedded human breast cancers taken from a population of Mexican women enriched for basal-like cancers show altered RNF8 and Ki67 mRNA expression compared to median expression as reference value. Results: Copy number differences in tumors for Ki67 and RNF8 mRNA were observed. The average differences in copy number for Ki67 between low and high expressing tumors was 8.8 fold and for RNF8 the average copy number differences between high and low expressors were 5.5 fold. When we compared the relationship between RNF8 and Ki67 expression within individual tumors, RNF8 expression was positively associated with Ki67 gene expression (p=0.04). Tumors that were high RNF8 were significantly more likely to also be high for Ki67. We found a non-significant but similar direction between RNF8 and cyclin B1 (p=0.08) but no relationship between RFN8 and cyclin D1 gene expression (p=0.318). This suggests that RNF8 is overexpressed in the tumors that are undergoing high mitoses. Conclusion: This is the first report demonstrating differential expression of RNF8 in a series of human breast cancers and a positive association between Ki67 and RNF8 levels. This finding is counter to our hypothesis that RNF8 acts as a tumor suppressor and that we would observe loss of gene expression. Interestingly, a recent study found that among BRCA deficient backgrounds, the involvement of an alternative DNA repair pathway that is prone to more mistakes involving p53 binding protein (53BP1), which interacts with RNF8 in cells, modifies susceptibility of the BRCA1 deficient background to carcinogenesis. Our findings may suggest that among some breast tumors overexpression of this altered DNA repair pathway including activation of RNF8 made contribute to the genomic instability in more aggressive tumors. Additional studies to assess the relationship between 53BP1 and RNF8 in these tumors are ongoing. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B47.

Early Detection of Response to Experimental Chemotherapeutic Top216 with [18F]FLT and [18F]FDG PET in Human Ovary Cancer Xenografts in Mice

Background3′-deoxy-3′-[18F]fluorothymidine (18F-FLT) is a tracer used to assess cell proliferation in vivo. The aim of the study was to use 18F-FLT positron emission tomography (PET) to study treatment responses to a new anti-cancer compound. To do so, we studied early anti-proliferative effects of the experimental chemotherapy Top216 non-invasively by PET.Methodology/Principal Findings In vivo uptake of 18F-FLT in human ovary cancer xenografts in mice (A2780) was studied at various time points after Top216 treatment (50 mg/kg i.v. at 0 and 48 hours) was initiated. Baseline 18F-FLT scans were made before either Top216 (n = 7–10) or vehicle (n = 5–7) was injected and repeated after 2 and 6 hours and 1 and 5 days of treatment. A parallel study was made with 2′-deoxy-2′-[18F]fluoro-D-glucose (18F-FDG) (n = 8). Tracer uptake was quantified using small animal PET/CT. Imaging results were validated by tumor volume changes and gene-expression of Ki67 and TK1. Top216 (50 mg/kg 0 and 48 hours) inhibited the growth of the A2780 tumor compared to the control group (P<0.001). 18F-FLT uptake decreased significantly at 2 hours (−52%; P<0.001), 6 hours (−49%; P = 0.002) and Day 1 (−47%; P<0.001) after Top216 treatment. At Day 5 18F-FLT uptake was comparable to uptake in the control group. Uptake of 18F-FLT was unchanged in the control group during the experiment. In the treatment group, uptake of 18F-FDG was significantly decreased at 6 hours (−21%; P = 0.003), Day 1 (−29%; P<0.001) and Day 5 (−19%; P = 0.05) compared to baseline.Conclusions/SignificanceOne injection with Top216 initiated a fast and significant decrease in cell-proliferation assessable by 18F-FLT after 2 hours. The early reductions in tumor cell proliferation preceded changes in tumor size. Our data indicate that 18F-FLT PET is promising for the early non-invasive assessment of chemotherapy effects in both drug development and for tailoring therapy in patients.

Increased Cellular Proliferation in Rat Skeletal Muscle and Tendon in Response to Exercise: Use of FLT and PET/CT

The purpose of this study is to investigate exercise-induced cellular proliferation in rat skeletal muscle/tendon with the use of 3'-[F-18]fluoro-3'deoxythymidine (FLT) and to quantitatively study concomitant changes in the proliferation-associated factor, Ki67. Wistar rats (n = 13) performed 3 days of treadmill running. Cellular proliferation was investigated 3 days before and 48 h after the running exercise with the use of FLT and positron emission tomography/computed tomography (PET/CT). Results were compared to a sedentary control group (n = 10). Image-derived standardized uptake values were calculated for Achilles tendons and calf muscles and compared to gene expression and immunohistochemical evaluations of Ki67. Treadmill running induced increased uptake of FLT uptake in calf muscles (30%; p < 0.001) and in Achilles tendon (21%, p < 0.001). The image-derived results were supported by a correlation in calf muscle to Ki67 (protein and mRNA level), while this coherence was not found in tendon. FLT-PET seems to be a promising tool for imaging of exercise-induced cellular proliferation in musculo-tendinous tissue.

Synergism of hydroxyapatite nanoparticles and recombinant mutant human tumour necrosis factor-α in chemotherapy of multidrug-resistant hepatocellular carcinoma

Locoregional chemotherapy continues to be the mainstay for the treatment of unresectable hepatocellular carcinoma (HCC). One of the principal obstacles implicated in its unsuccessful therapy is multidrug resistance (MDR). Former studies have identified the multidrug-resistant nature and possible mechanisms of hepatoma cells both in vitro and in vivo. This work aimed to develop an effective strategy for the treatment of HCC with MDR. The treatment was exploited to inhibit the MDR cells by co-administration of the recombinant mutant human tumour necrosis factor-alpha (rmhTNF-alpha), a sublethal dose of chemicals [adriamycin (ADM), mitomycin and 5-FU] and hydroxyapatite nanoparticles (nHAPs). Real-time quantitative reverse transcriptase-polymerase chain reaction and electrochemiluminescence Western blot were used to detect the expression of several related genes. The chemicals acted synergistically with rmhTNF-alpha and nHAP in suppressing the growth of hepatoma cells and inducing apoptosis of the cells, with the MDR phenotype reversed, as measured by intracellular ADM retention. Analysis of mRNA and protein revealed that rmhTNF-alpha inhibited the gene expression of XIAP, survivin, Ki67, PCNA, MDR1 and BCRP to some extent. Moreover, the inhibitory effects mentioned above could be as good or better than when nHAP is incorporated into the regimens. rmhTNF-alpha was not only able to restore the chemotherapeutic sensitivity to HepG2/ADM, its xenograft model and clinical samples but also further inhibited the growth of these tumours by a combination of nHAP. These results strongly suggested that chemicals in combination with rmhTNF-alpha and nHAP may be beneficial for the local treatment of advanced HCC.

Interactive effects of low-dose estradiol and tamoxifen on the postmenopausal primate endometrium and mammary gland.

Abstract Abstract #3047 Background: Long-term use of combined estrogen+progestin hormone therapies increases breast cancer risk in postmenopausal women. Selective estrogen receptor modulators (SERMs) are promising alternatives to progestins, particularly for breast cancer survivors and other women at high-risk for breast cancer considering hormone therapy. The purpose of this study was to evaluate interactive effects of low-dose estradiol (E2) and the SERM tamoxifen on the postmenopausal endometrium and mammary gland.&amp;#x2028; Materials and Methods: Ovariectomized adult female cynomolgus monkeys were given placebo (n=6), oral estradiol (E2, 0.25 mg/1800 kcal) (n=6), tamoxifen (TAM, 20 mg/1800 kcal) (n=5), or E2+TAM (n=6) for 4 months. Doses of E2 and TAM were scaled to approximate those given to women.&amp;#x2028; Results: Endometrial thickness and uterine weight were significantly higher in E2, TAM, and E2+TAM groups compared to placebo (P &amp;lt; 0.01 for all). In contrast, endometrial gland and stromal proliferation determined by Ki67 immunolabeling was significantly greater only in the E2 group (P &amp;lt; 0.01 for both). Endometrial Ki67 gene expression was also higher only in the E2 group (P &amp;lt; 0.01). Gene markers of estrogen receptor alpha (ER) activity such as TFF1 and STC2 were significantly increased in E2, TAM, and E2+TAM groups (P &amp;lt; 0.01 for all). On endometrial histology TAM and E2+TAM induced stromal fibrosis and occasional simple glandular hyperplasia with cystic change. In the mammary gland, TFF1 gene expression was increased by all treatments (P &amp;lt; 0.05 for all), while no statistically significant treatment effects were seen for epithelial proliferation or progesterone receptor expression.&amp;#x2028; Discussion: Previous studies have suggested that the weak estrogen-like effects of tamoxifen in the uterus contribute to increased endometrial cancer risk. Our findings reveal a more complex pattern of tamoxifen action involving increased ER activity, antagonism of E2-induced proliferation, and distinctive stromal remodeling. These results suggest that tamoxifen does not exhibit simple weak estrogen agonist / antagonist effects in the primate endometrium but instead acts through pathways independent of classical ER signaling to alter cancer risk. Future gene profiling should aid in the identification of these alternative pathways. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3047.

Prognostic Value of Gene Signatures and Tumorbiological Characteristics in Breast Cancer Patients Treated with Anthracycline-containing Chemotherapy

BACKGROUND: Many prognostic and predictive multigene signatures have been established in breast cancer patients. For treatment decision the assessment of individual prognosis is essential. The choice of specific therapy is basically driven by empirical data although several predictive gene signatures already exist. In this context it would be valuable if specific signatures could be used for estimation of prognosis and prediction of therapy concurrently. MATERIAL AND METHODS: Microarray data (Affymetrix HG U133A) of a small samples set of n = 48 breast cancer patients who received an anthracycline-based adjuvant chemotherapy were analyzed. Tumor samples were classified according to four prognostic and two predictive previously described gene signatures and compared with standard parameters as histologic subtype, tumor size, nodal status, pathohistological grading as well as estrogen receptor and Her-2 status. RESULTS: The gene expression values of ER, PR and Her-2 from microarray revealed a high concordance with protein expression assessed by means of immunohistochemistry. The determination of proliferative state of the tumors using gene expression of Ki67 showed a significant correlation with ER-status (p = 0.040, Mann-Whitney U-test) and pathohistological grading (p = 0.005, Kruskal-Wallis test). Neither of the six different signatures was able to predict event status of patients sufficiently. The main discriminatory power of the signatures was related to the ER status and to some extent to pathohistological grading. CONCLUSION: In a small cohort of uniformly treated patients prognostic and predictive gene signatures are incapable to predict disease outcome unambiguously. The main driving force of all signatures are ER-status and proliferation. The value of the individual signatures may be restricted to the specific setting from which they were derived.

Effects of estradiol with micronized progesterone or medroxyprogesterone acetate on risk markers for breast cancer in postmenopausal monkeys

The addition of the synthetic progestin medroxyprogesterone acetate (MPA) to postmenopausal estrogen therapy significantly increases breast cancer risk. Whether this adverse effect is specific to MPA or characteristic of all progestogens is not known. The goal of this study was to compare the effects of oral estradiol (E2) given with either MPA or micronized progesterone (P4) on risk biomarkers for breast cancer in a postmenopausal primate model. For this randomized crossover trial, twenty-six ovariectomized adult female cynomolgus macaques were divided into social groups and rotated randomly through the following treatments (expressed as equivalent doses for women): (1) placebo; (2) E2 (1 mg/day); (3) E2 + P4 (200 mg/day); and (4) E2 + MPA (2.5 mg/day). Hormones were administered orally, and all animals were individually dosed. Treatments lasted two months and were separated by a one-month washout period. The main outcome measure was breast epithelial proliferation, as measured by Ki67 expression. Compared to placebo, E2 + MPA resulted in significantly greater breast proliferation in lobular (P < 0.01) and ductal (P < 0.01) epithelium, while E2 + P4 did not. Intramammary gene expression of the proliferation markers Ki67 and cyclin B1 was also higher after treatment with E2 + MPA (P < 0.01) but not E2 + P4. Both progestogens significantly attenuated E2 effects on body weight, endometrium, and the TFF1 marker of estrogen receptor activity in the breast. These findings suggest that oral micronized progesterone has a more favorable effect on risk biomarkers for postmenopausal breast cancer than medroxyprogesterone acetate.

Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids

Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the Docetaxel group.

Apoptosis, proliferation and p53, cyclin D1, and retinoblastoma gene expression in relation to radiation response in transitional cell carcinoma of the bladder

Purpose: To determine whether the apoptotic index, the Ki67 index, and the expression of the p53, cyclin D1, and retinoblastoma genes correlate with local control, overall survival, and time to distant metastases in invasive bladder cancer treated with external beam radiation. Methods and Materials: Paraffin-embedded pretreatment biopsies from 83 patients with invasive transitional cell carcinoma of the bladder were scored morphologically for apoptosis and immunohistochemically for Ki67, p53, cyclin D1, and retinoblastoma gene expression. Survival analysis methods were used to assess overall survival, local control, and freedom from distant metastases. A multiple proportional hazard (PH) regression analysis was performed to study the prognostic value of the abovementioned biologic parameters (all divided into two categories, except Ki67) in addition to classical prognostic factors such as T stage, histologic grade, multifocality of the tumor, and completeness of transurethral resection. All patients were treated with external beam radiation as sole treatment. Median follow-up for the 19 patients still living was 7.5 years. Results: Apoptotic index varied from 0% to 3.4% with a mean of 0.8% and a median of 0.6%. Ki67 index varied from 0% to 60% with a mean of 14% and a median of 12%. P53 protein was detectable in 61% of the tumors. Overexpression of cyclin D1 was observed in 39% of the tumors and loss of retinoblastoma protein in 23% of the tumors. High Ki67 index was found to be significantly associated with p53 expression ( p = 0.04) and cyclin D1 overexpression ( p = 0.023). Cyclin D1 overexpression was found more often in Rb-positive tumors than in Rb-negative tumors ( p = 0.006). Other associations between the markers are less clear. Biologic markers were not correlated with T stage or grade. In the PH analysis local control was found to be significantly better for tumors with wild-type p53 ( p = 0.028). Also, tumors with an apoptotic index above the median value (0.6%) had a significantly better local control rate ( p = 0.035). Ki67 index ( p = 0.35), retinoblastoma gene expression ( p = 0.30) and cyclin D1 overexpression ( p = 0.61) were not found to have an additional predictive value regarding local tumor control. None of the tested biologic parameters were found to be associated with overall survival. Time to distant metastases was significantly shorter for tumors with high Ki67 index ( p = 0.01) and tumors with an apoptotic index less than median ( p = 0.009). Conclusions: The results of our study provide evidence for a prognostic value of p53 expression and apoptotic index with respect to the radiation response in bladder cancer in addition to more conventional prognosticators. The value of these parameters as a predictive assay for radiation response warrants confirmation in larger and prospective studies.

P21WAF1/CIP1 expression in gestational trophoblastic disease: correlation with clinicopathological parameters, and Ki67 and p53 gene expression.

BACKGROUND: The p21WAF1/CIP1 gene mediates growth arrest by inhibiting G1 cyclin dependent kinases and has been considered as a downstream effector of the tumour suppressor gene p53. AIM: To analyse...