DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.Eturauhassyöpä on miesten yleisimpiä syöpätyyppejä. Sen kehittyminen vaatii mieshormonialtistusta ja DNA-vaurioiden aiheuttamia muutoksia useissa solun kasvua säätelevien reittien toiminnassa. Viimeaikaisten tutkimusten mukaan virheet DNA:n kaksoissäiekatkosten korjauksessa voivat johtaa eturauhassyövässä tyypillisten geneettisten muutosten syntyyn. Ymmärrys näihin muutoksiin johtavista mekanismeista solussa auttaa eturauhassyövän hoidon kehittämisessä ja syövän ennaltaehkäisyssä. Eturauhanen ja seminaalivesikkelit kuuluvat miehen lisääntymiselimistöön, ja ne ovat rakenteellisesti ja toiminnallisesti samankaltaisia. Seminaalivesikkelikudoksen syöpä on kuitenkin äärimmäisen harvinainen, ja maailmanlaajuisesti on julkaistu vain noin 50 tapausta. Tässä työssä selvitimme, miten normaali eturauhanen ja seminaalivesikkeli tunnistavat DNA-vaurioita. Tätä tarkoitusta varten pystytimme uudenlaiset eturauhas- ja seminaalivesikkelikudosleikkeiden viljelymenetelmät, joihin aiheutimme DNA-vauriota säteilytyksellä tai kemikaaleilla. Näin pystyimme tutkimaan erilaistuneiden solutyyppien DNA-vauriovasteita. DNA:n kaksoissäiekatkokset käynnistivät tärkeän vauriontunnistusreitin molempien kudosten kaikissa pääsolutyypeissä. Yllättäen osa vauriosignaalia välittävistä proteiineista ei kuitenkaan toiminut samalla tavalla molempien kudosten kahdessa eri epiteelisolutyypissä. Lisäksi halusimme tutkia, miten eturauhasen ja seminaalivesikkelin epiteelisolut vastaavat DNA:n kaksoissäiekatkoksiin silloin kun ne jakautuvat aktiivisesti. Kudosleikemallissa jakautuvia soluja on vähän, joten selvitimme tätä kysymystä eturauhas- ja seminaalivesikkeliepiteelin primaarisolumalleissa. Primaarisolut eristetään potilasnäytteistä ja kasvatetaan solumaljoilla. Tietojemme mukaan tässä tutkimuksessa käytettiin ensimmäistä kertaa ihmisen seminaalivesikkeliepiteelin primaarisoluja. Aiheutimme sekä eturauhas- että seminaalivesikkelisoluihin kaksoissäiekatkoksia säteilytyksellä. Vastoin odotuksia havaitsimme, että eturauhasen epiteelisoluissa DNA-vaurio ei hidasta solunjakautumista, kun taas seminaalivesikkeliepiteelisoluissa solunjakautumisen hidastusvaste toimii. Tätä eroa voi selittää se, että seminaalivesikkelien epiteelisoluissa kaksi solunjakautumista DNA-vaurion jälkeen säätelevää proteiinia, p53 ja Wee1A, toimivat aktiivisemmin. Löydöksiemme perusteella on mahdollista, että eturauhasepiteeli on erityisen altis DNA-vaurioiden virheelliselle korjaukselle silloin, kun epiteelisolukko uudistuu, esimerkiksi tulehduksen aiheuttaman epiteelivaurion jälkeen

Long-term results of surgical resection of lung metastases from soft tissue sarcoma : A single center experience

Background A single-institution experience of pulmonary metastasectomy in soft tissue sarcoma (STS) was retrospectively reviewed. Our specific aim was to examine, whether the resection of pulmonary metastases could be curative. We also compared overall survival (OS) of patients after complete or incomplete pulmonary resection and nonsurgical treatment. Methods Between 1987 and 2016, 1580 patients were treated for STS with curative intent by Soft Tissue Sarcoma Group at Helsinki University Hospital, Finland. Three hundred forty-seven patients (22%) developed advanced disease and 130 STS patients (9%) developed pulmonary metastases as first systemic relapse. Seventy four patients (5%) were operated for lung metastases. Results Fifty-five patients (42%) had a complete and 19 (15%) incomplete resection. Fifty-six (43%) were unoperated. Median OS after complete or incomplete metastasectomy, chemotherapy, or best supportive care was 22, 18, 8, and 5 months, respectively. Twelve patients (9%) developed no further metastases and are alive with no evidence of disease. Disease-free survival (DFS) for completely resected patients was 17% at 5 years. All long-term survivors had oligometastatic disease and they underwent one to three complete metastasectomies. Conclusions Complete pulmonary metastasectomy in STS results in 5 years DFS in nearly one-fifth of patients. Most of these patients are probably cured.Peer reviewe

Viral Oncogene–Induced DNA Damage Response Is Activated in Kaposi Sarcoma Tumorigenesis

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)–infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers

Local relapse of soft tissue sarcoma of the extremities or trunk wall operated on with wide margins without radiation therapy

Background: The quality of surgical margins is the most important factor affecting local control in soft tissue sarcoma (STS). Despite this, there is no universally accepted consensus on the definition of an adequate surgical margin or on which patients should be offered radiation therapy. This study focuses on local control and its prognostic factors in patients with trunk wall and extremity STS. Methods: Adult patients with a final diagnosis of trunk wall or extremity STS referred to a single tertiary referral centre between August 1987 and December 2016 were identified from a prospective institutional database. Patients were treated according to a protocol instituted in 1987. The classification of surgical margins and indications for radiation therapy were based on anatomy and strict definition of surgical margins as metric distance to the resection border. Local treatment was defined as adequate if patients received either surgery with wide margins alone or marginal surgery combined with radiation therapy. Margins were considered wide if the tumour was excised with pathological margins greater than 2.5 cm or with an uninvolved natural anatomical barrier. After treatment, patients were followed up with local imaging and chest X-ray: 5 years for high-grade STS, 10 years for low-grade STS. Results: A total of 812 patients were included with a median follow-up of 5.8 (range 0.5-19.5) years. Forty-four patients had a grade 1 tumour: there were no instances of recurrence in this group thus they were excluded from further analysis. Five-year local control in the 768 patients with grade 2-3 STS was 90.1 per cent in patients receiving adequate local treatment according to the protocol. Altogether, 333 patients (43.4 per cent) were treated with wide surgery alone and their 5-year local control rate was 91.1 per cent. Among patients treated with wide surgery alone, deep location was the only factor adversely associated with local relapse risk in multivariable analysis; 5-year local control was 95.3 per cent in superficial and 88.3 per cent in deep-sited sarcomas (hazards ratio 3.154 (95% c.i. 1.265 to 7.860), P = 0.014). Conclusion: A high local control rate is achievable with surgery alone for a substantial proportion of patients with STS of the extremities or superficial trunk wall.Peer reviewe

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

Peer reviewe

Immunohistochemical analysis of Ad5 and Ad5T122 infection in ex vivo tissue cultures of normal human liver and colorectal carcinoma liver metastasis.

<p>Precision-cut tumour (top and third row of panels) and liver (second row and bottom panels) tissue cultures were left uninfected (left panels) or infected with 10⁷ PFU (in 2 ml of media) of Ad5 (middle panels) or Ad5T122 (right panels), and fixed five days later for staining with haematoxylin-eosin (panels G-L) or an antibody against the viral E1A protein (panels A–F). Necrosis developing in the central area of the uninfected control tissue (panel J) is indicated with arrows.</p

Comparison of cell killing by Ad5 and Ad5T122 in a panel of cancer cell lines.

<p>Four cell lines of non-hepatic (HCT116, A549, and Hep-2) or hepatic (Huh7) origin were infected with Ad5Luc1 (non-replicative virus control), Ad5, or Ad5T122 at an MOI of 0.05. Cell survival in the infected cells was measured 7 days (Hep-2 and A549) or 9 days (HCT116 and Huh7) post-infection using an ATP-based cell viability assay, and plotted on the y-axis as the percentage of the control values measured from uninfected cultures. The data are presented as the mean of six repetitions ± standard error.</p