RT-PCR analysis of the CDT2 and INTS7 genes in various human (A), mouse (B), and rat (C) tissues and cell lines.

<p>The lower panels in each part show the G3PDH bands of the ethidium bromide-stained gels as a control. The source of the cDNA is indicated at the top. RT-PCR products were derived from amplifications in the log range.</p

<i> In silico</i> analysis of the CDT2-INTS7 intergenic region.

<p>(A) Nucleotide sequence of the intergenic region of CDT2 and INTS7 genes. The sequences of seven mammalian genes are aligned and conserved nucleotides are marked with asterisks under the alignment. The bent arrows indicate the transcription start sites and direction of human genes (5′-CGATA— and 5′-AGCGC— for CDT2 and INTS7, respectively), canine genes, (5′-TCAGT— and 5′-AACAG— for CDT2 and INTS7, respectively), and mouse genes (5′-GGCGG— and 5′-CGCGG— for CDT2 and INTS7, respectively). The bent arrows positioned on the sequences are for CDT2. The bent arrows positioned under the sequences are for INTS7. Transfac software (threshold >80) predicts four E2F consensus sites (E2F A∼D, marked with dotted boxes), and Sp1 (5′-GAGGCGGGGA), NF-Y (5′-AAGCCAATCAG), CREB (5′-TGACGTCA), and Myb (5′-CCAAACTGAC) transcription factor-binding sites (marked by arrows with dotted lines). (B) Computer predicted threshold (Transfac software) of E2F A∼D were summarized for seven mammalian genes.</p

Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.

<p>(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, <i>P</i><0.05 for pcDNA3 versus E2F1).</p

Promoter analysis of the CDT2-INTS7 intergenic region.

<p>(A) The structure of human, mouse, and canine CDT2 and INTS7 genes in the head-to-head orientation. Translation start codons (represented by ATG) of the CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate the E2F consensus sites (threshold >85). Arrows with numbers were the region and direction used for the luciferase (Luc) assay. (B) Luciferase expression of pGL3-human −363/+1, pGL3-mouse −335/+32, and pGL3-canine −312/+54 constructs in A549 cells are shown as fold induction with respect to the pGL3-Basic vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3). (C) Luciferase expression of pGL3-human −363/+1 (hereafter denoted as ABCD), pGL3-mouse −335/+32, and pGL3-canine −312/+54 constructs in A549 cells were up-regulated by co-expressing the E2F1, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3).</p

The human CDT2-INTS7 intergenic region acts as a bidirectional promoter.

<p>(A) Structure of human CDT2 and INTS7 genes in the head-to-head orientation. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) Luciferase expression of pGL3 constructs are summarized in (A) in A549 cells and are shown as fold induction with respect to the pGL3-Basic vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3).</p

IGF-I enhanced p53-p21 pathway in human fibroblasts.

<p><b>A, B,</b> p53 and p21 mRNA expression in human fibroblasts treated with GH or IGF-I for 27 days (PDL of 20–21). The expressions were measured using quantitative RT-PCR and normalized to β-actin. <b>C,</b> Immunoblotting analysis of p53, serine-phosphorylated-p53 (p-p53), and p21 proteins in human fibroblasts treated with GH or IGF-I for 27 days (PDL of 20–21). Anti-p-p53 protein at serine 15 antibody was used for the detection of p-p53. <b>D, E,</b> Densitometric analysis of p53, p-p53, and p21 protein levels normalized to β-actin. All data are expressed as mean ± standard deviation. Data were compared using one-way analysis of variance followed by post hoc Fisher’s least significant difference test. *<i>P</i> < 0.05, **<i>P</i> < 0.01; PDL, population doubling levels.</p

Telomere length in human fibroblasts with GH and IGF-I treatment.

<p><b>A, B,</b> Longitudinal changes in relative telomere length in human fibroblasts treated with GH and IGF-I. <b>C, D,</b> Telomere shortening rate in human fibroblasts treated with GH and IGF-I. All data are expressed as mean ± standard deviation. Data were compared using one-way analysis of variance followed by post hoc Fisher’s least significant difference test. *<i>P</i> < 0.05, **<i>P</i> < 0.01. PDL, population doubling levels; RTL, relative telomere length.</p

Clinical characteristics of the acromegaly (Acro) and the non-functioning pituitary adenoma (NFPA) group.

<p>Data were compared by the χ² test, Fischer’s exact test, Student’s <i>t</i>-test, or Mann—Whitney test, as appropriate.</p><p>**<i>P</i> < 0.01.</p><p>RTL, relative telomere length; Acro, acromegaly; NFPA, non-functioning pituitary adenoma; E2, estradiol; T, testosterone. E2 and T were measured in female and male, respectively.</p><p>Clinical characteristics of the acromegaly (Acro) and the non-functioning pituitary adenoma (NFPA) group.</p

Telomere length in patients with acromegaly.

<p><b>A,</b> Comparison of relative telomere length between patients with acromegaly and non-functioning pituitary adenoma. <b>B–H</b>, Correlations between relative telomere length and clinical indices (age, BMI, disease duration, tumor diameter, tandom GH, nadir GH during OGTT, and IGF-I SDS). <b>I</b>, Comparison of relative telomere length for the clinical indices (sex, hypertension, diabetes, dyslipidemia, macroadenoma) in patients with acromegaly. Relative telomere length was compared using Student’s <i>t</i>-tests. Data are expressed as median with interquartile range. *<i>P</i> < 0.05, **<i>P</i> < 0.01. RTL, relative telomere length; Acro, acromegaly; NFPA, non-functioning pituitary adenoma.</p

IGF-I induced cellular senescence in human fibroblasts.

<p><b>A,</b> Senescence-associated β-galactosidase staining in human fibroblasts treated with GH or IGF-I for 38 days (PDL of 28–29). <b>B, C,</b> Rate of senescence-associated β-galactosidase activity (SA β-gal)-positive cells. <b>D, E,</b> Cell growth curves of human fibroblasts treated with GH or IGF-I. <b>F,</b> Proposed mechanistic model, in which excess secretion of GH and IGF-I causes increased comorbidities and mortality in patients with acromegaly. Data are expressed as mean ± standard deviation. Data were compared using χ² test followed by Tukey’s honestly significant difference test. **<i>P</i> < 0.01; PDL, population doubling levels.</p