Validating reference-based algorithms to determine cell-type heterogeneity in ovarian cancer DNA methylation studies.

Information about cell composition in tissue samples is crucial for biomarker discovery and prognosis. Specifically, cancer tissue samples present challenges in deconvolution studies due to mutations and genetic rearrangements. Here, we optimized a robust, DNA methylation-based protocol, to be used for deconvolution of ovarian cancer samples. We compared several state-of-the-art methods (HEpiDISH, MethylCIBERSORT and ARIC) and validated the proposed protocol in an in-silico mixture and in an external dataset containing samples from ovarian cancer patients and controls. The deconvolution protocol we eventually implemented is based on MethylCIBERSORT. Comparing deconvolution methods, we paid close attention to the role of a reference panel. We postulate that a possibly high number of samples (in our case: 247) should be used when building a reference panel to ensure robustness and to compensate for biological and technical variation between samples. Subsequently, we tested the performance of the validated protocol in our own study cohort, consisting of 72 patients with malignant and benign ovarian disease as well as in five external cohorts. In conclusion, we refined and validated a reference-based algorithm to determine cell type composition of ovarian cancer tissue samples to be used in cancer biology studies in larger cohorts.

Laser-assisted topical delivery of vismodegib reduces hedgehog gene expression in human basal cell carcinomas in vivo.

BACKGROUND: Systemically delivered hedgehog inhibitors including vismodegib and sonidegib are widely used to treat basal cell carcinomas (BCCs). Ablative fractional laser (AFL)-assisted topical delivery of vismodegib has been demonstrated in preclinical studies. The aim of this explorative clinical study was to evaluate intratumoral vismodegib concentrations and effect on hedgehog pathway gene expression following AFL-assisted topical vismodegib delivery to BCCs. METHODS: In an open-label clinical trial, 16 nodular BCCs (in n = 9 patients) received one application of CO2 -AFL (40 mJ/microbeam, 10% density) followed by topical vismodegib emulsion. After 3-4 days, vismodegib concentrations in tumor biopsies (n = 15) and plasma were analyzed and compared with samples from patients receiving oral treatment (n = 3). GLI1, GLI2, PTCH1, and PTCH2 expression was determined by quantitative polymerase chain reaction (n = 7) and GLI1 additionally by in situ hybridization (n = 3). RESULTS: Following AFL-assisted topical administration, vismodegib was detected in 14/15 BCCs and reached a median concentration of 6.2 µmol/L, which compared to concentrations in BCC tissue from patients receiving oral vismodegib (9.5 µmol/L, n = 3, p = 0.8588). Topical vismodegib reduced intratumoral GLI1 expression by 51%, GLI2 by 55%, PTCH1 and PTCH2 each by 73% (p ≤ 0.0304) regardless of vismodegib concentrations (p ≥ 0.3164). In situ hybridization demonstrated that GLI1 expression was restricted to tumor tissue and downregulated in response to vismodegib exposure. CONCLUSION: A single AFL-assisted topical application of vismodegib resulted in clinically relevant intratumoral drug concentrations and significant reductions in hedgehog pathway gene expressions.

Test of the FlashFREEZE unit in tissue samples freezing for biobanking purposes.

Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.

Prospects of Improving Early Ovarian Cancer Diagnosis Using Cervical Cell Swabs.

Ovarian cancer (OC) has the poorest prognosis and the highest mortality rate among gynecological malignancies, which is largely due to delayed diagnosis. Therefore, an effective detection strategy is a compelling need. Here, we review the potential use of cervical cell swabs (Pap specimens, liquid) for early detection of OC. It has been shown, that malignant cells exfoliate from the ovaries and may be detected in Pap specimens, routinely collected through cervical cancer screening. Using Medical Subject Headings (MeSH) for searching the PubMed database we identified eight studies reporting the use of Pap specimen in early detection of OC. Six focused on detection of gene mutations, using gene panels or analysis of TP53 variants. Two studies reported analysis of methylation profiles. Seven studies were published in 2018 or later. Additionally, we found one study without MeSH terms assigned yet, which postulated using peptide biomarkers present in Pap-test fluid. In this review we present their main findings, discuss challenges this approach presents and include ideas for improved detection.

Cell membrane permeability and defective G2/M block as factors potentially contributing to increased cell chemosensitivity. SeAx cell line as an example.

BACKGROUND: Immortalized mammalian cell lines are a valuable research tool, though they represent a highly simplified model. Due to accumulated mutations they may not reflect characteristics of the disease or even the tissue they derive from. OBJECTIVE: We aim to pinpoint factors distinguishing SeAx cells from two other cutaneous T-cell lymphoma (CTCL) cell lines, namely Hut78 and MyLa2000. Of note, these factors may influence cell sensitivity in an unspecific way and therefore should be taken under consideration. METHODS: We evaluated transcriptional levels of drug transporters across cell lines, cell membrane permeability, functionality of pathways related to DNA damage response and activation of G2/M block. RESULTS: Analysis of the transcriptional levels of genes coding drug efflux pumps indicated that they are not consistently down-regulated in SeAx. However, we noted that SeAx cell membrane is markedly more permeable than Hut78 and MyLa2000, which may contribute to increased chemosensitivity in an unspecific way.Moreover, though DNA damage response seemed to be at least partly functional in SeAx cells, they fail to activate G2/M block in response to psoralen + UVA treatment. Any DNA damage should be repaired before cells enter mitosis, in order to uphold genome integrity. Thus, a defective cell cycle block may contribute to cell sensitivity. CONCLUSIONS: We believe that factors such as increased membrane permeability or defective cell cycle block should be accounted for when comparing sensitivity of cell line panels to chemotherapeutics of interest. It is worth to exclude a simple, indiscriminative mechanisms of cell resistance or sensitivity before attempting comparisons. Cell lines that are indiscriminately sensitive to a broad range of chemicals may contribute to overestimating the cytotoxic potential of tested compounds if used in cytotoxicity studies.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-ß1 Treatment on Collagen Deposition and Biomarkers.

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-ß1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-ß1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-ß1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-ß1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-ß1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

Electronic Pneumatic Injection-Assisted Dermal Drug Delivery Visualized by Ex Vivo Confocal Microscopy.

BACKGROUND AND OBJECTIVES: Electronic pneumatic injection (EPI) is a technique for dermal drug delivery, which is increasingly being used in clinical practice. However, only few studies have been reported on cutaneous drug distribution and related clinical endpoints. We aimed to visualize the immediate cutaneous drug distribution, changes in skin architecture, and related clinical endpoint of EPI. STUDY DESIGN/MATERIALS AND METHODS: Acridine orange (AO) solution was administered to ex vivo porcine skin by EPI at pressure levels from 4 to 6 bar with a fixed injection volume of 50 µl and nozzle size of 200 µm. Immediate cutaneous distribution was visualized using ex vivo confocal microscopy (EVCM). Changes in skin architecture were visualized using both EVCM and hematoxylin and eosin-stained cryosections. RESULTS: The defined immediate endpoint was a clinically visible papule formation on the skin. The pressure threshold to consistently induce a papule was 4 bar, achieving delivery of AO to the deep dermis (2319 µm axial and 5944 µm lateral distribution). Increasing the pressure level to 6 bar did not lead to significant differences in axial and lateral dispersion (P = 0.842, P = 0.905; respectively). A distinctively hemispherical distribution pattern was identified. Disruption of skin architecture occurred independently of pressure level, and consisted of subepidermal clefts, dermal vacuoles, and fragmented collagen. CONCLUSIONS: This is the first study to relate a reproducible clinical endpoint to EPI-assisted immediate drug delivery using EVCM. An EPI-induced skin papule indicates dermal drug delivery throughout all layers of the dermis, independent of pressure level settings. Lasers Surg. Med. © 2020 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Photochemotherapy Induces Interferon Type III Expression via STING Pathway.

DNA-damaging cancer therapies induce interferon expression and stimulate the immune system, promoting therapy responses. The immune-activating STING (Stimulator of Interferon Genes) pathway is induced when DNA or double-stranded RNA (dsRNA) is detected in the cell cytoplasm, which can be caused by viral infection or by DNA damage following chemo- or radiotherapy. Here, we investigated the responses of cutaneous T-cell lymphoma (CTCL) cells to the clinically applied DNA crosslinking photochemotherapy (combination of 8-methoxypsoralen and UVA light; 8-MOP + UVA). We showed that this treatment evokes interferon expression and that the type III interferon IFNL1 is the major cytokine induced. IFNL1 upregulation is dependent on STING and on the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS). Furthermore, 8-MOP + UVA treatment induced the expression of genes in pathways involved in response to the tumor necrosis factor, innate immune system and acute inflammatory response. Notably, a subset of these genes was under control of the STING-IFNL1 pathway. In conclusion, our data connected DNA damage with immune system activation via the STING pathway and contributed to a better understanding of the effectiveness of photochemotherapy.

Psoralen with ultraviolet A-induced apoptosis of cutaneous lymphoma cell lines is augmented by type I interferons via the JAK1-STAT1 pathway.

BACKGROUND: Photochemotherapy with psoralen and ultraviolet A (PUVA), with or without adjuvant interferon-α (IFN-α), is a first-line therapy for early-stage mycosis fungoides and other forms of cutaneous T-cell lymphoma (CTCL). However, the mechanism by which PUVA with IFN-α work in CTCL is poorly understood. PURPOSE: To develop a model to investigate the mechanisms of PUVA and PUVA with IFN-α in CTCL cells. METHODS: An in vitro model to study the molecular mechanisms of PUVA was created using two different CTCL cell lines, MyLa, which has functional p53, and HuT-78, in which p53 is inactivated due to a homozygous nonsense mutation. RESULTS: PUVA caused G2/M cell cycle block and apoptosis of MyLa and HuT-78 accompanied by increase in the expression of the mitochondrial pro-apoptotic genes Bax, BAK, and PUMA and a downregulation in anti-apoptotic Bcl-2. p53 was induced and c-Myc was repressed by PUVA, but neither were essential for PUVA-induced apoptosis. IFN-α augmented PUVA-induced apoptosis via the JAK1 pathway, and this activity could be inhibited by ruxolitinib. CONCLUSION: PUVA induces p53-independent apoptosis in CTCL cell lines, and this process is augmented by type I interferons via the JAK1 pathway.

Chemotherapeutic treatment is associated with Notch1 induction in cutaneous T-cell lymphoma.

The Notch pathway is important for survival of cutaneous T-cell lymphoma (CTCL) cells. We investigated the effect of chemotherapy (doxorubicin, etoposide, and gemcitabine) and radiation modalities on Notch signaling in CTCL cell lines. Chemotherapy induced Notch1 expression at the mRNA and protein level in MyLa2000 and Hut78. Upregulation of well-established Notch targets supported the functional activity of Notch1. Transfection of Notch1 siRNA into MyLa2000 cells was not able to suppress the effects of chemotherapy on Notch1 activation significantly. Notch1 knockdown in combination with doxorubicin, etoposide, or gemcitabine compared to chemotherapy alone decreased cell viability by 12, 20, and 26%, respectively (p < 0.05). Additionally, X-rays (in MyLa2000 but not SeAx) and psoralen plus UVA (PUVA) (in MyLa2000, Hut78, and SeAx) increased the expression of Notch1 family members. Our results indicate that CTCL cells activate the Notch pathway in vitro in response to chemotherapy and radiation modalities as a possible protective mechanism.

Small-molecule inhibitors of Ataxia Telangiectasia and Rad3 related kinase (ATR) sensitize lymphoma cells to UVA radiation.

BACKGROUND: Psoralen plus ultraviolet A (PUVA) photochemotherapy is a combination treatment used for inflammatory and neoplastic skin diseases such as mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL). However, 30% of MF patients do not respond sufficiently to PUVA and require more aggressive therapies. OBJECTIVE: The aim of this project was to investigate whether inhibition of Ataxia Telangiectasia and Rad3 related kinase (ATR) may enhance efficacy of phototherapy. METHODS: CTCL cell lines (MyLa2000, SeAx and Mac2a) served as in vitro cell models. ATR and Chk1 were inhibited by small molecule antagonists VE-821, VE-822 or Chir-124, or by small interfering RNAs (siRNAs). Cell cycle and viability were assessed by flow cytometry. RESULTS: Small molecule inhibitors of ATR and Chk1 potently sensitized all cell lines to PUVA and, importantly, also to UVA, which by itself did not cause apoptotic response. VE-821/2 blocked ATR pathway activation and released the cells from the G2/M block caused by UVA and PUVA, but did not affect apoptosis caused by other chemotherapeutics (etoposide, gemcitabine, doxorubicine) or by hydrogen peroxide. Knockdown of ATR and Chk1 with siRNA also blocked the ATR pathway and released the cells from G2/M block but did not sensitize the cells to UVA as observed with the small molecule inhibitors. The latter suggested that the synergism between VE-821/2 or Chir-124 and UVA was not solely caused by specific blocking of ATR kinase but also ATR-independent photosensitization. This hypothesis was further verified by administrating VE-821/2 or Chir-124 before and after UVA irradiation, as well as comparing their activity with other ATR and Chk1 inhibitors (AZD6738 and MK8776). We found that only VE-821/2 and Chir-124 kinase inhibitors had synergistic effect with UVA, and only if applied before treatment with UVA. CONCLUSION: Small molecule ATR and Chk1 inhibitors potently sensitize lymphoma cells to UVA radiation and induce a prominent apoptotic response. Interestingly, this effect is due to the dual (kinase inhibiting and photosensitizing) mode of action of these compounds.

Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma.

Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53-/-). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL.

Favourable results of Mohs micrographic surgery for basal cell carcinoma.

INTRODUCTION: Basal cell carcinoma (BCC) is the most common malignant neoplasm with an annual incidence approaching 200/100,000 person-years. Mohs micrographic surgery (MMS) is widely used in North America and in Europe for treatment of BCC. This technique ensures radical tumour removal, sparing of the surrounding healthy skin, and it also offers higher cure rates than standard tumour excision with a predefined margin of healthy skin. The superiority of MMS relies on the fact that the entire (100%) margin of the excised tissue is examined microscopically for residual tumour in contrast to the traditional histopathological examination, in which 2% of the margin is examined. METHODS: In Denmark, MMS was first introduced by us in 2012. In the present study, we retrospectively included all patients who underwent MMS from May 2012 to June 2015. RESULTS: A total of 231 patients with 263 BCC were included. The mean age was 66.1 years. The most common localisations were the forehead (31.3%), the nose (31.0%) and the cheek (14.7%). Primary BCC comprised 54.0%; the remaining cases were relapses, most frequently after curettage (36.9%), radiotherapy (18.9%) and photodynamic therapy (11.7%). MMS leads to 40% smaller skin defects than standard excisions with 4 or 6 mm margins. Closure of skin defects was achieved by side-to-side closure in 49% and by local flaps in 40%. There were no relapses during the observation time. The safety, cosmetic and functional outcome were excellent. CONCLUSIONS: We recommend that MMS be included in the Danish BCC treatment guidelines, especially for high-risk BCC in the face, in line with standard practice in Europe and the United States. FUNDING: none. TRIAL REGISTRATION: not relevant.

Ellipticine induces apoptosis in T-cell lymphoma via oxidative DNA damage.

The tumor suppressor p53 is often mutated in human cancers. Restoring its antitumor activity has been shown to be a promising therapeutic approach for cancer treatment. Here we analyzed the activity and mechanism of a p53 reactivator, ellipticine, in a cellular model of cutaneous T-cell lymphoma (CTCL), a disease that is progressive, chemoresistant and refractory to treatment. We tested the effect of ellipticine in three cell lines with different p53 status: MyLa2000 (p53(wt/wt)), SeAx ((G245S)p53) and Hut-78 ((R196Stop)p53). Ellipticine caused apoptosis in MyLa2000 and SeAx and restored the transcriptional activity of (G245S)p53 in SeAx. However, p53 siRNA knockdown experiments revealed that p53 was not required for ellipticine-induced apoptosis in CTCL. The lipophilic antioxidant α-tocopherol inhibited ellipticine-dependent apoptosis and we linked the apoptotic response to the oxidative DNA damage. Our results provide evidence that ellipticine-induced apoptosis is exerted through DNA damage and does not require p53 activation in T-cell lymphoma.

miR-125b induces cellular senescence in malignant melanoma.

BACKGROUND: Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory. METHODS: We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and ß-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization. RESULTS: In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours. CONCLUSIONS: Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.

The importance of Notch signaling in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTLs) represent an area of high medical need. Previously, we demonstrated high expression of Notch, a known oncogene, in primary cutaneous anaplastic large cell lymphoma (ALCL). In this study, we performed immunohistochemical staining for Notch1 in lymph nodes from PTL not otherwise specified (PTL-NOS) and systemic ALCL (ALK+ and ALK-) and report a similar distribution among the three subgroups: Negative, moderate and strong expression was, respectively, 18%, 27% and 55% for PTL-NOS (33 cases), 20%, 0% and 80% for ALCL ALK+ (10 cases) and 45%, 22% and 33% for ALCL ALK- (nine cases) (p > 0.05). In the ALK+ ALCL cell line, Karpas-299, pharmacological inhibition of Notch with γ-secretase inhibitor (GSI) I was far more potent than with GSI IX, XX and XXI with regard to cell viability and apoptosis. In conclusion, PTL tumor cells have prominent Notch1 expression and treatment with Notch inhibitors has cytotoxic effects.

cMyc/miR-125b-5p signalling determines sensitivity to bortezomib in preclinical model of cutaneous T-cell lymphomas.

Successful/effective cancer therapy in low grade lymphoma is often hampered by cell resistance to anti-neoplastic agents. The crucial mechanisms responsible for this phenomenon are poorly understood. Overcoming resistance of tumor cells to anticancer agents, such as proteasome inhibitors, could improve their clinical efficacy. Using cutaneous T-cell lymphoma (CTCL) as a model of the chemotherapy-resistant peripheral lymphoid malignancy, we demonstrated that resistance to proteasome inhibition involved a signaling between the oncogene cMyc and miR-125b-5p. Bortezomib repressed cMyc and simultaneously induced miR-125b-5p that exerted a cytoprotective effect through the downmodulation of MAD4. Overexpression of cMyc repressed miR-125b-5p transcription and sensitized lymphoma cells to bortezomib. The central role of miR-125b-5p was further confirmed in a mouse model of T-cell lymphoma, where xenotransplantation of human CTCL cells overexpressing miR-125b-5p resulted in enhanced tumor growth and a shorter median survival. Our findings describe a novel mechanism through which miR-125b-5p not only regulates tumor growth in vivo, but also increases cellular resistance to proteasome inhibitors via modulation of MAD4.

Triterpenoid α-amyrin stimulates proliferation of human keratinocytes but does not protect them against UVB damage.

Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (HaCaT) by about 18% while other extract components were ineffective. A panel of biochemical and cell-based assays testing the antioxidative and cytoprotective activites of α-amyrin indicated no antioxidative activity of this compound. α-Amyrin did not protect HaCaT cells against the damage caused by UVB radiation.

MDM2 inhibitor nutlin-3a induces apoptosis and senescence in cutaneous T-cell lymphoma: role of p53.

P53 is rarely mutated in cutaneous T-cell lymphoma (CTCL) and is therefore a promising target for innovative therapeutic approaches. Nutlin-3a is an inhibitor of MDM2 (human homolog of murine double minute 2), which disrupts its interaction with p53, leading to the stabilization and activation of p53. To investigate the potential therapeutic use of nutlin-3a in CTCL, we screened CTCL lines Hut-78, SeAx, MyLa2000, Mac1, and Mac2a by measuring p53 levels after nutlin-3a treatment. In MyLa2000, Mac1, and Mac2a, we observed the increase in p53, indicating the fully functional p53. In the remaining cell lines, P53 mutation analysis identified a homozygous nonsense mutation (R196Stop in Hut-78) and a homozygous missense mutation (G245S in SeAx). In MyLa2000, Mac1, and Mac2a carrying wild-type P53, nutlin-3a induced apoptosis and senescence demonstrated by permanent G0/G1 cell-cycle block and expression of the senescence-associated ß-galactosidase. This effect was abolished in cells in which p53 was silenced by small interfering RNA. Sézary cells lack functional p53 and were resistant to nutlin-3a. However, nutlin-3a potentiated the efficacy of conventional chemotherapeutics not only in cells with intact p53 but also in Hut-78, SeAx, and Sézary cells. Thus, targeting p53 by nutlin-3a may constitute a therapeutic approach in CTCL because of increased apoptosis and senescence of tumor cells.

No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts.

The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-ß1 (TGF-ß1), which is presumed to be reduced systemically with advanced age. The aim of this study was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-ß1 levels in serum obtained from young and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells.