Hubert Stöppler

Recent prostate-specific antigen-based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression.

Three naturally occurring variant human papillomavirus type 16 (HPV-16) E6 proteins, which contained amino acid substitutions predominantly near the N terminus, exhibited significant differences in their abilities to abrogate keratinocyte differentiation in response to serum and calcium and to induce the degradation of p53 in vitro. One variant surpassed the reference E6 protein in its ability to abrogate keratinocyte differentiation responses, whereas another showed a reduction in this activity. Interestingly, the biological activities of the HPV-16 E6 proteins and their abilities to induce p53 degradation in vitro were directly correlated. These results demonstrate that naturally occurring variants of HPV-16 differ in biological and biochemical properties which might result in differences in pathogenicity.

The “high risk” subgroup of human papillomaviruses (e.g. HPV-16 and HPV-18) infect and induce tumors of mucosal epithelium. These neoplasms, which can progress to malignancy, retain and express the papillomavirus E6 andE7 oncogenes. In vitro, the E6 and E7 proteins associate with the cellular p53 and Rb proteins and interfere with their normal growth-regulatory functions. We report here that primary human keratinocytes transduced with the HPV-16 E6 gene, but not the E7 gene, express significant telomerase activity. However, despite this detectable enzymatic activity,E6-transduced cells continue to shorten their telomeres during in vitro passaging similar to control cells and to cells expressing the E7 and E6+E7 genes. At late passages, however, E7-transduced cells partially restore telomere length, although they lack detectable telomerase activity, demonstrating that E6-independent, telomerase-independent events mediate this change. The “high risk” subgroup of human papillomaviruses (e.g. HPV-16 and HPV-18) infect and induce tumors of mucosal epithelium. These neoplasms, which can progress to malignancy, retain and express the papillomavirus E6 andE7 oncogenes. In vitro, the E6 and E7 proteins associate with the cellular p53 and Rb proteins and interfere with their normal growth-regulatory functions. We report here that primary human keratinocytes transduced with the HPV-16 E6 gene, but not the E7 gene, express significant telomerase activity. However, despite this detectable enzymatic activity,E6-transduced cells continue to shorten their telomeres during in vitro passaging similar to control cells and to cells expressing the E7 and E6+E7 genes. At late passages, however, E7-transduced cells partially restore telomere length, although they lack detectable telomerase activity, demonstrating that E6-independent, telomerase-independent events mediate this change. The human papillomaviruses (HPVs) 1The abbreviations used are: HPV, human papillomavirus; TRAP; telomeric amplification protocol; kb, kilobase(s). 1The abbreviations used are: HPV, human papillomavirus; TRAP; telomeric amplification protocol; kb, kilobase(s).associated with cervical cancer are designated as the “high risk” subgroup of HPVs (e.g. HPV-16 and 18) (1zur Hausen H. Virology. 1991; 184: 9-13Crossref PubMed Scopus (857) Google Scholar) and encode two viral oncogenes, E6 and E7, which exhibit immortalizing and transforming activities in various rodent and human cell types (2–14; reviewed in Refs. 15Mansur C.P. Androphy E.J. Biochem. Biophys. Acta. 1993; 1155: 323-345PubMed Google Scholar and 16Stöppler H. Conrad Stöppler M. Schlegel R. Intervirology. 1994; 37: 168-179Crossref PubMed Scopus (26) Google Scholar). The oncogenic potential of these proteins is due, at least in part, to their ability to interfere with the function of two cellular tumor suppressor proteins, p53 and the retinoblastoma susceptibility gene product, Rb. The E6 protein binds to the cellular p53 protein and promotes its ubiquitin-dependent degradation (17Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3444) Google Scholar, 18Werness B.A. Levine A.J. Howley P.M. Science. 1990; 248: 76-79Crossref PubMed Scopus (2158) Google Scholar). E7 protein associates with Rb and interferes with its binding to E2F, resulting in impaired Rb cell cycle control functions (19Heck D.V. Yee C.L. Howley P.M. Münger K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4442-4446Crossref PubMed Scopus (297) Google Scholar, 20Dyson N. Howley P.M. Münger K. Harlow E. Science. 1989; 243: 934-937Crossref PubMed Scopus (2388) Google Scholar, 21Dyson N. Guida P. Münger K. Harlow E. J. Virol. 1992; 66: 6893-6902Crossref PubMed Google Scholar). The natural host cell of these viruses, the keratinocyte, is immortalized by the efficient expression of E7 protein (22Halbert C.L. Demers G.W. Galloway D.A. J. Virol. 1991; 65: 473-478Crossref PubMed Google Scholar). Although the E6 protein cannot independently immortalize these cells, it greatly augments the biological activity of the E7 protein (23Barbosa M.S. Schlegel R. Oncogene. 1989; 4: 1529-1532PubMed Google Scholar), induces resistance to signals for terminal differentiation (24Sherman L. Schlegel R. J. Virol. 1996; 70: 3269-3279Crossref PubMed Google Scholar), and prolongs precrisis life span (25Klingelhutz A.J. Barber S.A. Smith P.P. Dyer K. McDougall J.K. Mol. Cell. Biol. 1994; 14: 961-969Crossref PubMed Scopus (176) Google Scholar). Recently, it has been demonstrated that the expression ofE6in primary human keratinocytes leads to an activation of telomerase (26Klingelhutz A.J. Foster S.A. McDougall J.K. Nature. 1996; 380: 79-82Crossref PubMed Scopus (699) Google Scholar), an enzyme capable of preventing the shortening of telomeres during DNA replication (27Blackburn E.H. Szostak J.W. Annu. Rev. Biochem. 1984; 53: 163-194Crossref PubMed Scopus (506) Google Scholar, 28Blackburn E.H. Nature. 1991; 350: 569-573Crossref PubMed Scopus (3042) Google Scholar, 29Lindsey J. McGill N.I. Lindsey L.A. Green D.K. Cooke H.J. Mutat. Res. 1991; 256: 45-48Crossref PubMed Scopus (407) Google Scholar, 30Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1082) Google Scholar, 31Zakian V.A. Annu. Rev. Genet. 1989; 23: 579-604Crossref PubMed Scopus (530) Google Scholar). Telomeres, the ends of human eucaryotic chromosomes, are shortened progressively during cell aging in vivo and in vitro (29Lindsey J. McGill N.I. Lindsey L.A. Green D.K. Cooke H.J. Mutat. Res. 1991; 256: 45-48Crossref PubMed Scopus (407) Google Scholar, 30Harley C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1082) Google Scholar, 32Harley C.B. Futcher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4587) Google Scholar, 33Hastie N.D. Dempster M. Dunlop M.G. Thompson A.M. Green D.K. Allshire R.C. Nature. 1990; 346: 866-868Crossref PubMed Scopus (1471) Google Scholar, 34Allsopp R.C. Vaziri H. Patterson C. Goldstein S. Younglai E.V. Futcher A.B. Greider C.W. Harley C.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10114-10118Crossref PubMed Scopus (1942) Google Scholar). The telomeric ends of chromosomes consist of a stretch of tandemly repeated sequences complexed with DNA-binding proteins that position chromosomes within the nucleus, protect chromosomal ends, and prevent chromosomal fusion that can occur in late-passage or senescent cells (27Blackburn E.H. Szostak J.W. Annu. Rev. Biochem. 1984; 53: 163-194Crossref PubMed Scopus (506) Google Scholar, 28Blackburn E.H. Nature. 1991; 350: 569-573Crossref PubMed Scopus (3042) Google Scholar, 31Zakian V.A. Annu. Rev. Genet. 1989; 23: 579-604Crossref PubMed Scopus (530) Google Scholar). Germ-cell telomeres are longer than somatic cell telomeres and are maintained with age (35Allshire R.C. Dempster M. Hastie N.D. Nucleic Acids Res. 1989; 17: 4611-4627Crossref PubMed Scopus (317) Google Scholar, 36Cross S.H. Allshire R.C. McKay S.J. McGill N.I. Cooke H.J. Nature. 1989; 338: 771-774Crossref PubMed Scopus (119) Google Scholar, 37de Lange T. Shiue L. Myers R.M. Cox D.R. Naylor S.L. Killery A.M. Varmus H.E. Mol. Cell. Biol. 1990; 10: 518-527Crossref PubMed Scopus (713) Google Scholar), probably due to the activated riboprotein telomerase in these cells (38Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L.C. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.L. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6515) Google Scholar). In contrast to primary somatic cells, immortalized cells generally retain telomere length during in vitropropagation. The DNA polymerase telomerase, a ribonucleoprotein complex, is capable of elongating the 3′ lagging DNA strand by adding tandemly repeated sequences to this DNA strand. The ribonucleoprotein complex uses parts of its RNA molecule as a template for the polymerization of telomeric tandem repeats, counteracting the shortening of telomeres during the replication of DNA by DNA polymerase α. We observed that early-passage keratinocytes contained detectable telomerase activity that was lost at later passage numbers (between passages 2 and 6). Because both E6 and E7 genes can enhance and prolong human primary keratinocyte growth in vitro, we evaluated whether they might also induce alterations in chromosomal processing and maintain telomeres in a lengthened state. The analysis of primary human foreskin keratinocytes expressing the HPV-16 E6, E7, or E6+E7 (E6/7) genes revealed that E6- and E6/7-expressing cells were telomerase-positive, independent of their passage number.E7-expressing cells, like vector-infected control cells, demonstrated an activated telomerase at early-passage numbers that was rapidly lost during cell passaging. The detectable telomerase activity in precrisis E6- and E6/7-expressing cells, however, did not result in the maintenance of telomere length. All cells, independent of their expression of HPV oncogenes and telomerase, equally shortened their telomeres. However, E7-expressing cells demonstrated partial restoration of telomeric length without the concomitant activation of telomerase during their extended life span. Primary human keratinocytes were derived from neonatal foreskins as described (10Schlegel R. Phelps W.C. Zhang Y.L. Barbosa M. EMBO J. 1988; 7: 3181-3187Crossref PubMed Scopus (193) Google Scholar) and grown in KSF medium (Life Technologies, Inc.) supplemented with gentamycin. The primary cells were infected with derivatives of the amphotrophic LXSN retrovirus expressing the various HPV-16 open reading frames (E6, E7, and E6+E7). The retroviruses were generated as described (39Miller A.D. Rosman G.J. BioTechniques. 1989; 7: 980-990PubMed Google Scholar) using existing recombinant vectors (24Sherman L. Schlegel R. J. Virol. 1996; 70: 3269-3279Crossref PubMed Google Scholar). Retrovirus-infected cells were selected in G418 (100 μg/ml medium) for 10 days. G418-resistant colonies were pooled from each transduction and passaged every 3–4 days (ratio of 1:5). A modified telomeric amplification protocol (TRAP) was performed as described by Kim et al. (38Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L.C. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.L. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6515) Google Scholar). Cells were harvested by trypsinization followed by a wash in Dulbecco's modified Eagle's medium containing 10% fetal calf serum to inactivate trypsin. The cells were washed a second time in phosphate-buffered saline and frozen at −70 °C. Cell pellets (≈5 × 106) were lysed in 800 μl of lysis buffer (0.5% 3-((3-cholamidopropyl)-dimethyl-ammonio)-1-propanesulfonate, 10 mm Tris-Cl, pH 7.5, 1 mm MgCl2, 1 mm EGTA, 5 mm β-mercaptoethanol, and 10% glycerol) for 30 min on ice. The lysates were then centrifuged for 2 min at 14,000 × g (4 °C), and 600 μl of the supernatant were removed. The protein concentration of the supernatant was determined using a DC Protein Assay kit (Bio-Rad). RNase inhibitor (RNasin from Promega Corp.) was added afterward to a final concentration of 10 units/ml. The remaining nuclear pellet was digested with proteinase K and treated with phenol; chromosomal high molecular weight DNA was isolated by standard techniques for Southern blot analysis. The TRAP assay was performed in a 0.2-ml MicroAmp reaction tube (Perkin Elmer) containing 98 μl of a reaction mixture composed of 1 × Taq polymerase buffer (10 mmTris-HCl, pH 8.8, 1 mm KCl, and 0.02% Tween 20), 1.5 mm MgCl2, 100 ng of TS primer (5′-AATCCGTCGAGCAGAGTT-3′), 200 μm of each deoxynucleoside triphosphate, and 0.05–6 μg of protein extract. The reaction mixture was incubated for 20 min at 25 °C. The samples were then heated to 80 °C, and 1 μl (100 ng/μl) of CX primer (3′-AATCCCATTCCCATTCCCATTCCC-5′) and 0.5 μl of Taqpolymerase (UlTima DNA polymerase, 6 units/μl; Perkin Elmer) were added before the samples were heated to 94 °C for 90 s. The polymerase chain reaction was performed for 28 cycles under the following conditions: 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s. Ten % of the polymerase chain reaction product was separated on 8% SDS-polyacrylamide gel (1 × TBE), and the gel was stained with a Gelcode color silver stain kit (Pierce). To demonstrate telomerase specificity, control samples were either digested for 20 min at 37 °C with 1 μg of RNase A and 1 μg of RNase H or the samples were heated for 10 min to 100 °C prior to the telomerase reaction. Three or 10 μg of chromosomal high molecular weight DNA were restricted with the indicated restriction endonucleases for telomere Southern blot analysis or HPV copy number analysis, respectively. The restricted DNA samples were separated on 1 × TAE agarose gels (0.5–1.0%) and blotted onto a nylon membrane. A 32P kinase-labeled primer, (TTAGGG)3, was used under standard high stringency hybridization conditions (T m −15 °C) to detect the telomeric ends. A random-primed 32P-labeled HPV-16E6/7 DNA fragment was used to detect the various HPV open reading frames using high stringency conditions. Primary human foreskin keratinocytes were infected with amphotropic retroviruses expressing either the HPV-16 E6, E7, E6/7 genes or the neomycin resistance gene (control). Cells were selected in serum-free keratinocyte medium containing G418 as described under “Experimental Procedures” and passaged at a ratio of 1:5. Keratinocytes infected with control retrovirus ceased proliferation at passages 10–12, whereas those transduced with E6/7 established into cell lines. Keratinocytes expressing either E6 or E7 alone exhibited extended life span and continued to proliferate beyond passage 26. We observed, in three independent experiments, thatE6 retroviruses generated human keratinocyte strains with significant telomerase activity at early passage (e.g.passages 4 and 6, Fig. 1 A), similar to the recently published results of Klingelhutz et al. (26Klingelhutz A.J. Foster S.A. McDougall J.K. Nature. 1996; 380: 79-82Crossref PubMed Scopus (699) Google Scholar). The concomitant expression of the E6/7 genes gave similar results. Although low levels of telomerase were observed in control keratinocytes and in E7-expressing cells at early passages (passages 4 and 6), this activity was absent at later passages (passages 8 and 10), presumably reflecting the loss of basal epithelial “stem” cells in the neonatal human foreskin keratinocyte population. The detection of telomerase activity in normal keratinocytes was dependent upon the isolation procedures and the number of cell divisions needed to establish the primary culture and was normally detected up to passages 2–3 but never after passage 6. Mixing experiments (Fig. 1 B) using a telomerase-negativeE7 cell extract (Fig. 1 A; E7 passage 8) and telomerase-positive cell extracts (Fig. 1 A; E6, E6/7 passage 8, and a cell extract from an HPV-16 immortalized keratinocyte line) demonstrated that the loss of TRAP assay activity inE7 extracts was not due to the presence of an inhibitor of telomerase or polymerase activity. Thus, the gradual passage-dependent loss of telomerase activity inE7-expressing cells represents a true loss of enzymatic activity. To determine whether the observed activation of telomerase in E6-transduced keratinocytes (Fig.1 A) was accompanied by altered processing of the chromosome telomeres, we harvested nuclei from keratinocytes at the indicated passages and performed Southern blotting on isolated cellular DNA with a telomeric probe to evaluate average telomere length (Fig.1 C). Cells were evaluated until passage 10 when the HPV-negative control cells reached crisis. Southern blot analysis demonstrated that regardless of the HPV genes transfected, all cells exhibited shortened telomeres similar to control-transduced cells. During the first 10 passages, the average telomere length decreased from approximately 10 to 8.5 kb, which represents an average loss of 60–80 base pairs per population doubling. This is in agreement with previous studies in primary human fibroblasts (32Harley C.B. Futcher A.B. Greider C.W. Nature. 1990; 345: 458-460Crossref PubMed Scopus (4587) Google Scholar, 34Allsopp R.C. Vaziri H. Patterson C. Goldstein S. Younglai E.V. Futcher A.B. Greider C.W. Harley C.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10114-10118Crossref PubMed Scopus (1942) Google Scholar) and human embryonic kidney cells. The correlation between the loss of keratinocyte growth potential (at 30–40 population and the shortening of telomeres by also the in telomere length in vivo between fibroblasts from human and C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1082) Google Scholar, 33Hastie N.D. Dempster M. Dunlop M.G. Thompson A.M. Green D.K. Allshire R.C. Nature. 1990; 346: 866-868Crossref PubMed Scopus (1471) Google Scholar). We also the telomerase activity of keratinocytes at passage the time when the HPV-negative control cells and lost their potential The TRAP assay in Fig. 2 A cells continued to telomerase activity whereas the and E7-transduced cells were The expression of the E6 or E7 protein leads to an extended keratinocyte in vitro in to or vector-infected control cells (25Klingelhutz A.J. Barber S.A. Smith P.P. Dyer K. McDougall J.K. Mol. Cell. Biol. 1994; 14: 961-969Crossref PubMed Scopus (176) Google Scholar). In of cells beyond passage 26. the continued presence of telomerase activity in these late-passage cells (Fig. 2 A), we observed a shortening of the telomere length to from at passage (Fig. 2 E6-transduced cells lost an 60–80 base pairs per population doubling. The cells also demonstrated a similar loss of telomeric length between passages 10 and (Fig. 2 However, from approximately passage numbers to keratinocytes an in telomere length (Fig. 2 although was detectable telomerase activity in these cells (Fig. 2 In cells at passage continued to shorten their telomeres. The in telomere length of E7-expressing cells might as observed in culture Greider C.W. Harley C.B. S. EMBO J. 1992; PubMed Scopus Google Scholar), that the cells reached crisis. Although all keratinocyte strains were isolated following G418 and consist of cells transduced with HPV we performed Southern blot analysis of chromosomal DNA from the indicated keratinocyte strains to the presence of viral genes. Keratinocytes transduced with E6, E7, or at passages 4 and 10 to determine the average copy number of HPV genes in the cell population. Fig. that all keratinocyte strains contained at least copy of the transduced HPV gene per cell at both early and late This result that the observed shortening of telomeres cells is not the of gene transduction or HPV gene that or all of the E6-transduced keratinocytes are expressing telomerase is derived from the analysis of telomerase activity in transduced cells and derived cell immortalized by either HPV-16 or DNA or by The telomerase activities of the cell were to the telomerase activity of cells not The extracts of transduced cell strains exhibited telomerase activity that was similar to that observed in independent cell that the transduced keratinocyte population the cell of cells expressing To demonstrate that the polymerase chain TRAP assay was telomerase activity than the of extracts with chromosomal telomeric we treated the cell extracts with RNase A and H for 20 min at 37 °C or with 100 °C for 10 In both telomerase activity was that the riboprotein telomerase was for the observed results of cell extracts demonstrated cells telomerase activity similar cells and that the of the TRAP assay was to detect activity in approximately 100 cells (Fig. 4 This is with the of the assay using established cell (e.g. previous that keratinocyte population was composed of cells expressing telomerase The ability of the E6 protein to the precrisis life span of human keratinocytes and to telomerase is in agreement with the independent of Klingelhutz et al. (26Klingelhutz A.J. Foster S.A. McDougall J.K. Nature. 1996; 380: 79-82Crossref PubMed Scopus (699) Google Scholar). However, by telomere length and telomerase activity during passages, we demonstrated that the activation of telomerase has detectable on the normal shortening of telomeres during in passaging. In we also demonstrated cells telomeres after passaging at cellular and that this telomere independent of detectable telomerase activity. The that E7-transduced cells telomeres without detectable telomerase activity is in agreement with a of et al. A. J. S. EMBO J. 14: PubMed Scopus Google Scholar) human cell without detectable telomerase activity. Although the of telomeres during and after cell normally with a high of chromosomal keratinocytes immortalized or chromosomal J.M. Schlegel R. 1994; PubMed Scopus Google Scholar, N. Res. 1990; Google Scholar). The that the in telomerase activity is and by the time the keratinocytes been selected in However, it is whether this in enzymatic activity represents the of telomerase, the activation of telomerase, or the of telomerase-positive cells. Thus, it a following the of primary keratinocytes by the E6 the E6 augments the growth of telomerase-positive cells. This result in the amplification of a keratinocyte population containing high telomerase activity. is also that all of the primary keratinocytes express telomerase activity and that E6 functions to maintain this However, it is that telomerase activity in not in somatic cells but is restricted to cells and tumor cells C.B. Mutat. Res. 1991; 256: 271-282Crossref PubMed Scopus (1082) Google Scholar, N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L.C. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.L. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6515) Google Scholar). The telomerase activity detected in E6-transduced keratinocytes is to the activity of all cells in the population than a for two Southern blot analysis on the each keratinocyte at least copy of the transduced HPV Thus, it is that of the transduced cells are expressing E6 and altered in telomerase activity. however, are in techniques to evaluate whether E6 protein is in each and the of telomerase detected in the E6-transduced keratinocyte population is to the telomerase observed in a keratinocyte cell that was immortalized by E6/7 (Fig. Although the E6 protein cellular telomerase activity, both E6 and E7 are to the keratinocyte life span in vitro, that of keratinocyte proliferation is not dependent upon telomerase activity. In that telomere length is not the for the of keratinocyte proliferation in vitro E6- and E7-transduced cells telomeres than control cells. results demonstrate that or telomerase activity is to induce cellular E6-transduced keratinocytes not establish into cell lines. The that E7-transduced cells telomeres after passaging that telomerase-independent for the of telomeres in E7-transduced cells. the of et al. A. J. S. EMBO J. 14: PubMed Scopus Google Scholar), that a for the of telomeres that is not accompanied by detectable levels of is that the observed of telomeres inE7-expressing cells to with the of cell a potential in cell We Conrad Stöppler for and reading of the

INTRODUCTION: Recent studies suggest that in addition to factors such as treatment side effects, suicide, and poor health habits, people with schizophrenia may have an increased risk of diabetes prior to antipsychotic treatment. Diabetes is associated with an increased pulse pressure (PP) and a shortened telomere. We tested the hypothesis that prior to antipsychotic treatment, schizophrenia and related disorders are associated with a shortened telomere, as well as an increased PP. METHODS: Telomere content (which is highly correlated with telomere length) and PP were measured in newly diagnosed, antipsychotic-naive patients with schizophrenia and related disorders on first clinical contact and in matched control subjects. Both groups were also administered an oral glucose tolerance test. RESULTS: Compared with control subjects, the patients with psychosis had decreased telomere content and an increased PP. As previously reported, they also had increased glucose concentrations at 2 hours. These differences could not be attributed to differences in age, ethnicity, smoking, gender, body mass index, neighborhood of residence, socioeconomic status, aerobic conditioning, or an increased cortisol concentration in the psychotic subjects. DISCUSSION: These results suggest that prior to antipsychotic use, nonaffective psychosis is associated with reduced telomere content and increased PP, indices that have been linked to an increased risk of diabetes and hypertension.