Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non-small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromosomal instability--evidence of E2F-1 transcriptional control over hCdt1 - PubMed (original) (raw)
Comparative Study
Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non-small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromosomal instability--evidence of E2F-1 transcriptional control over hCdt1
Panagiotis Karakaidos et al. Am J Pathol. 2004 Oct.
Abstract
Replication licensing ensures once per cell cycle replication and is essential for genome stability. Overexpression of two key licensing factors, Cdc6 and Cdt1, leads to overreplication and chromosomal instability (CIN) in lower eukaryotes and recently in human cell lines. In this report, we analyzed hCdt1, hCdc6, and hGeminin, the hCdt1 inhibitor expression, in a series of non-small-cell lung carcinomas, and investigated for putative relations with G(1)/S phase regulators, tumor kinetics, and ploidy. This is the first study of these fundamental licensing elements in primary human lung carcinomas. We herein demonstrate elevated levels (more than fourfold) of hCdt1 and hCdc6 in 43% and 50% of neoplasms, respectively, whereas aberrant expression of hGeminin was observed in 49% of cases (underexpression, 12%; overexpression, 37%). hCdt1 expression positively correlated with hCdc6 and E2F-1 levels (P = 0.001 and P = 0.048, respectively). Supportive of the observed link between E2F-1 and hCdt1, we provide evidence that E2F-1 up-regulates the hCdt1 promoter in cultured mammalian cells. Interestingly, hGeminin overexpression was statistically related to increased hCdt1 levels (P = 0.025). Regarding the kinetic and ploidy status of hCdt1- and/or hCdc6-overexpressing tumors, p53-mutant cases exhibited significantly increased tumor growth values (Growth Index; GI) and aneuploidy/CIN compared to those bearing intact p53 (P = 0.008 for GI, P = 0.001 for CIN). The significance of these results was underscored by the fact that the latter parameters were independent of p53 within the hCdt1-hCdc6 normally expressing cases. Cumulatively, the above suggest a synergistic effect between hCdt1-hCdc6 overexpression and mutant-p53 over tumor growth and CIN in non-small-cell lung carcinomas.
Figures
Figure 1-4261
mRNA and protein analysis of hCdt1 and hCdc6. A: Representative results of comparative RT-PCR (i) and Western blot analysis (ii) for hCdt1 in paired normal (N)**/tumor (T) samples; case 9 with overexpression, and cases 10 and 11 with normal expression of hCdt1. B: Representative results of comparative RT-PCR (i) and Western blot analysis (ii) for hCdc6 in paired normal (N)/**tumor (T) samples; cases 30 and 31 with overexpression and case 32 with normal expression of hCdc6. In A and B, the corresponding charts show the compared (as a tumor/normal ratio) and normalized (target to reference) cDNA and protein levels, respectively, of the matched tumor/normal samples. GAPDH mRNA and actin protein were used as reference for RT-PCR and Western blotting analysis, respectively.
Figure 2-4261
Scatter plot of the normalized tumor-to-normal cDNA and protein ratios calculated for the hCdt1 (A) and hCdc6 (B) expression of the examined NSCLCs. According to the hCdt1 and hCdc6 cDNA and protein expression, the carcinomas were subdivided in two groups: one consisted of cases characterized by overexpression at both mRNA and protein levels, whereas the other comprised tumors that exhibited mRNA and protein levels similar to those of their normal counterparts. The mean value and its SD of the corresponding ratios are presented under the arrows that indicate the values’ range for each subgroup. Scatter plots present the NSCLC subgroups according to hCdt1/hCdc6 patterns of the normalized protein (C) or tumor/normal cDNA (D) ratios.
Figure 3-4261
Representative immunohistochemical results of two cases harboring hCdc6 overexpression and one with normal expression (as scored by RT-PCR and Western blot analysis), alongside their matching normal counterparts (Ai, Bi, and Ci). Case 26 (squamous cell carcinoma) (Aii, Aiii) and case 27 (adenocarcinoma) (Bii, Biii) exhibit strong hCdc6 immunostaining, whereas case 69 (undifferentiated large cell carcinoma) (Cii, Ciii) displays normal hCdc6 immunoreactivity (very weak cytoplasmic staining) (showed by arrow in Ciii). As shown in the accompanying pictures of higher magnification (Aiii and Biii, respectively), the hCdc6 immunostaining was mainly cytoplasmic and occasionally nuclear (positive nuclei are demarcated by arrowheads). hCdc6 immunostaining was in accordance with Western blot analysis (Aiv, Biv, and Civ). D: Western blot analysis for hCdc6 on total, nuclear, and cytoplasmic protein extracts from case 26 verifying the results of the immunocytochemical analysis and implying that the applied hCdc6 antibody detects the cytoplasmic as well as the nuclear form of hCdc6.
Figure 4-4261
Representative results of E2F-1 analysis in two cases (cases 13 and 46) demonstrating high E2F-1 labeling index and a third one (case 18) displaying low E2F-1 expression levels. In case 13, E2F-1 overexpression was associated with inactivation of pRb (as revealed by p-pRb immunoreactivity in serial sections). In case 46, E2F-1 overexpression was accompanied by deregulated total pRb expression (absence of immunostaining that was associated with pRb LOH). For comparison, in case 18 the observed lower EI (as assessed also by Western blot analysis) was associated with a lower degree of p-pRb immunoreactivity. As described in the Materials and Methods section, the following assays are presented in the figure: for pRb status: IHC against total pRb and p-pRb, and LOH analysis using the D13S153 internal pRb microsatellite marker [the arrowhead indicates allelic loss as estimated by the densitometric values (presented in the corresponding boxes below the chart) based on previously described criteria32]; for E2F-1: IHC and Western blot analysis.
Figure 5-4261
i: Schematic representation of hCdt1 promoter with putative transcription factor binding sites indicated. The predicted transcription start site is shown by an arrow, whereas the 1067-nucleotide fragment used for further analysis is marked. ii: Differential regulation of the hCdt1 promoter by members of the E2F family. NIH3T3 cells were transfected with 1000 ng of the hCdt1 promoter cloned into pGL3 vector alone (Cdt1Prom) or in combination with 200 ng of vectors expressing the six members of the E2F family (+E2F 1 to 6) together with 500 ng of a β-gal-expressing plasmid as an internal control. Luciferase values, corrected for total protein and transfection efficiency, are expressed as fold increase over the activity exhibited by Cdt1Prom alone. Standard deviations resulted from three independent experiments, each performed in triplicates. iii: hCdt1 promoter activity in synchronized NIH3T3 cells. NIH3T3 cells were transfected with the reporter construct containing the hCdt1 promoter (Cdt1Prom) and subsequently cultured in 0.1% serum for 48 hours (0 hours). Cell proliferation was induced by addition of 10% calf serum and cells were harvested at the indicated time points (6, 12, 24 hours) to assay luciferase and galactosidase activity. Asynchronously growing cells were transfected with hCdt1 promoter containing plasmid and luciferase activity was measured (Cdt1 FM). In addition, an E2F1 expression plasmid enhances transcription from an hCdt1 promoter-driven luciferase construct on serum starvation when co-transfected in NIH3T3 cells (Cdt1 SF 0 hours and Cdt1 SF 0 hours +E2F1). Luciferase values of triplicate experiments are presented as fold increase of the promoter activity observed in serum-starved cells.
Figure 6-4261
Representative results showing downstream events associated with overexpression of hCdt1 and hCdc6 in two cases (cases 13 and 46). In case 13 bearing wild-type p53, low proliferation and high apoptotic indices were observed. On the other hand, in case 46 harboring mutant p53 (C135R), high proliferation and low apoptotic indices, as well as, CIN (aneuploidy) were recorded. As described in the Materials and Methods section the following assays are presented in the figure: for hCdt1: C-RT-PCR and Western blot analysis; for hCdc6: C-RT-PCR, Western blot and IHC analysis; for p53: IHC and sequencing analysis; for proliferation: IHC against Ki-67; for apoptosis: TUNEL assay (apoptotic nuclei are depicted by arrows); for CIN: ploidy analysis (the x axis represents nuclear DNA quantity in c units, whereas y axis corresponds to the number of evaluated nuclei).
Figure 7-4261
Survival curves of patients with deregulated hCdt1-hCdc6 stratified according to p53 status. In this subgroup of carcinomas with deregulated hCdt1-hCdc6, mutant p53, as compared to wild-type p53, tended to be associated with adverse prognosis (P = 0.08 by Kaplan-Meier methodology).
Figure 8-4261
i: Representative C-RT-PCR analysis of matched normal (N)-tumor (T) tissues from cases 21, 38, and 43, showing normal, underexpression, and overexpression of hGeminin, respectively. ii: Representative Western blot analysis of two paired N-T cases (cases 38 and 43), confirming the previously observed underexpression and overexpression of hGeminin by C-RT-PCR in i. iii: Bar chart depicting the percentage of tumors exhibiting normal (white bars) and overexpressed hCdt1 (black bars) when hGeminin was evaluated as underexpressed, normal, and overexpressed. iv: Bar chart depicting the percentage of aneuploid (black bars) and diploid (white bars) cases according to the p53/hGeminin patterns.
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