Core outcome sets for prevention and treatment of postpartum haemorrhage: an international Delphi consensus study.

OBJECTIVE: To develop core outcome sets (COS) for studies evaluating interventions for (1) prevention and (2) treatment of postpartum haemorrhage (PPH), and recommendations on how to report the COS. DESIGN: A two-round Delphi survey and face-to-face meeting. POPULATION: Healthcare professionals and women's representatives. METHODS: Outcomes were identified from systematic reviews of PPH studies and stakeholder consultation. Participants scored each outcome in the Delphi on a Likert scale between 1 (not important) and 9 (critically important). Results were discussed at the face-to-face meeting to agree the final COS. Consensus at the meeting was defined as ≥ 70% of participants scoring the outcome as critically important (7-9). Lectures, discussion and voting were used to agree how to report COS outcomes. MAIN OUTCOME MEASURES: Outcomes from systematic reviews and consultations. RESULTS: Both Delphi rounds were completed by 152/205 (74%) participants for prevention and 143/197 (73%) for treatment. For prevention of PPH, nine core outcomes were selected: blood loss, shock, maternal death, use of additional uterotonics, blood transfusion, transfer for higher level of care, women's sense of wellbeing, acceptability and satisfaction with the intervention, breastfeeding, and adverse effects. For treatment of PPH, 12 core outcomes were selected: blood loss, shock, coagulopathy, hysterectomy, organ dysfunction, maternal death, blood transfusion, use of additional haemostatic intervention, transfer for higher level of care, women's sense of wellbeing, acceptability and satisfaction with the intervention, breastfeeding, and adverse effects. Recommendations were developed on how to report these outcomes where possible. CONCLUSIONS: These COS will help standardise outcome reporting in PPH trials. TWEETABLE ABSTRACT: Core outcome sets for PPH: nine core outcomes for PPH prevention and 12 core outcomes for PPH treatment.

Phytochemical Characterization, Antibacterial, Acetylcholinesterase Inhibitory and Cytotoxic Properties of Cryptostephanus vansonii, an Endemic Amaryllid.

Cryptostephanus vansonii I. Verd., an endemic Amaryllidaceae species from Zimbabwe, was evaluated for its acetylcholinesterase (AChE) inhibitory and cytotoxicity properties using Ellman's colorimetric method and the tetrazolium-based colorimetric assay against Vero monkey kidney cells, respectively. The plant extracts were also evaluated for their antibacterial activity against five bacteria. Furthermore, phytochemical profiles of the extracts were determined using ultra-high performance liquid chromatography coupled with tandem mass spectrometry analysis. A plant part-dependent AChE inhibitory activity was observed, in the order, root > rhizome > basal leaf > leaf. Overall, C. vansonii extracts exhibited better antibacterial activity against Gram-negative compared with Gram-positive bacteria. Cytotoxic effects were not detected in Vero monkey kidney cell lines suggesting the possible absence of toxophores in C. vansonii extracts. Similar to the trend in biological activity, a distinct plant part-dependent variation in hydroxybenzoates, hydroxycinnamates and flavonoids was observed in the plant extracts. In addition, 5-hydroxymetylfurfural and eucomic acid were detected in the different plant parts of C. vansonii. The results of the present study provide valuable AChE inhibition activity, toxicological and phytochemical profiles of C. vansonii. Further studies on isolation of bioactive compounds and their subsequent evaluation in other pharmacological and toxicological model systems are required. Copyright © 2017 John Wiley & Sons, Ltd.

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo.

In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg-Prkdc(scid)IL2rγ(tm1Wjl) (abbreviated NOD-scid IL2rγ(null)) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the ß-globin gene using nanoparticles carrying ß-globin-targeted PNAs/DNAs, demonstrating this method's versatility. In vivo testing in an enhanced green fluorescent protein-ß-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

Production of a severe cystic fibrosis mutation in mice by gene targeting.

We have used gene targeting in embryonic stem cells to introduce an HPRT mini-gene into the coding sequence of the murine cystic fibrosis gene (cftr). This insertion introduces a termination codon in frame with the cftr coding sequence to terminate prematurely the CFTR protein within the first nucleotide binding domain. Animals homozygous for the cftr disruption fail to thrive and display a range of symptoms including meconium ileus, distal intestinal obstructions, gastrointestinal mucus accumulation and blockage of pancreatic ducts. The animals also show lacrimal gland pathology. Tracheal and caecal transepithelial current measurements demonstrate the lack of a cAMP activatable Cl- channel. These animals will prove useful for the evaluation of new therapeutic drugs and gene therapy strategies.

Generation and characterization of a delta F508 cystic fibrosis mouse model.

We have generated mice carrying the most common mutation in cystic fibrosis (CF), delta F508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. delta F508-/- mice die from peritonitis and show deficiencies in cAMP-activated electrogenic Cl- transport. These mice produce delta F508 transcripts and show the temperature-dependent trafficking defect first described for the human delta F508 CFTR protein. A functional CFTR Cl- channel not demonstrated by null CF mice or present at 37 degrees C was detected following incubation of epithelial cells at 27 degrees C. Thus, these mice are an accurate delta F508 model and will be valuable for testing drugs aimed at overcoming the delta F508 trafficking defect.

SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome.

Leigh Syndrome (LS) is a severe neurological disorder characterized by bilaterally symmetrical necrotic lesions in subcortical brain regions that is commonly associated with systemic cytochrome c oxidase (COX) deficiency. COX deficiency is an autosomal recessive trait and most patients belong to a single genetic complementation group. DNA sequence analysis of the genes encoding the structural subunits of the COX complex has failed to identify a pathogenic mutation. Using microcell-mediated chromosome transfer, we mapped the gene defect in this disorder to chromosome 9q34 by complementation of the respiratory chain deficiency in patient fibroblasts. Analysis of a candidate gene (SURF1) of unknown function revealed several mutations, all of which predict a truncated protein. These data suggest a role for SURF1 in the biogenesis of the COX complex and define a new class of gene defects causing human neurodegenerative disease.

Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice.

Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero-injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (Cftr(tmlCam)) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr(-/-) mice treated with the CFTR-adenovirus and those treated with the control vector.

Immature reticulocyte fraction as a useful parameter for blood transfusion assessment in anaemia.

During erythropoietic stress (e.g., acute anaemia) the reticulocyte count in peripheral blood normally increases as the bone marrow responds to increased erythropoietin stimulation of erythroid precursors. The efficiency of this process is an indicator of the patient's bone marrow response. This study assesses the utility of the immature reticulocyte fraction (IRF) as a useful parameter of anaemia type, which may inform the decision to treat with red cell transfusion. Moreover, it investigates the value of using IRF as an inexpensive, non-invasive and objective indicator of a patient's bone marrow response. EDTA-treated venous blood specimens were collected from in-patients with a haemoglobin value <100 g/L and analysed to establish the absolute reticulocyte count and IRF using the ABX Pentra 120 Retic analyser. Based on the clinical information provided, the specimens were divided into those with chronic anaemia and those with acute anaemia. Statistical analysis of results showed that there was a significant negative correlation between IRF and haemoglobin level. Importantly, IRF was also found to show a more significant correlation with haemoglobin level than did the absolute reticulocyte count. Furthermore, this correlation was stronger in patients with acute versus chronic anaemia. Thus, this information may aid clinicians in their decisions to recommend blood transfusions for patients with certain types of anaemia.

Practice variation in total hip arthroplasty versus hemiarthroplasty for treatment of fractured neck of femur in Australia.

AIMS: Displaced femoral neck fractures (FNF) may be treated with partial (hemiarthroplasty, HA) or total hip arthroplasty (THA), with recent recommendations advising that THA be used in community-ambulant patients. This study aims to determine the association between the proportion of FNF treated with THA and year of surgery, day of the week, surgeon practice, and private versus public hospitals, adjusting for known confounders. PATIENTS AND METHODS: Data from 67 620 patients in the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) from 1999 to 2016 inclusive were used to generate unadjusted and adjusted analyses of the associations between patient, time, surgeon and institution factors, and the proportion of FNF treated with THA. RESULTS: Overall, THA was used in 23.7% of patients. THA was more frequently used over time, in younger patients, in healthier patients, in cases performed on weekdays (adjusted odds ratio (OR) 1.27; 95% confidence interval (CI) 1.14 to 1.41), in private hospitals (adjusted OR 4.34; 95% CI 3.94 to 4.79) and by surgeons whose hip arthroplasty practice has a relatively higher proportion of elective patients (adjusted OR 1.65; 95% CI 1.49 to 1.83). CONCLUSION: Practice variation exists in the proportion of FNF patients treated with THA due to variables other than patient factors. This may reflect variation in resources available and surgeon preference, and uncertainty regarding the relative indication.

Non-obese diabetic-recombination activating gene-1 (NOD-Rag1 null) interleukin (IL)-2 receptor common gamma chain (IL2r gamma null) null mice: a radioresistant model for human lymphohaematopoietic engraftment.

Immunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non-obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene-1 (Rag1(null)) and interleukin (IL)-2 receptor common gamma chain (IL2rgamma(null)), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17-Prkdc(scid) (NOD-Prkdc(scid)) IL2rgamma(null) mice. We observed that immunodeficient NOD-Rag1(null) IL2rgamma(null) mice tolerated much higher levels of irradiation conditioning than did NOD-Prkdc(scid) IL2rgamma(null) mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation-conditioned adult mice, leading to multi-lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1(null) and scid stocks of immunodeficient NOD mice harbouring the IL2rgamma(null) mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD-Rag1(null) IL2rgamma(null) mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.

Chloride transporting capability of Calu-3 epithelia following persistent knockdown of the cystic fibrosis transmembrane conductance regulator, CFTR.

BACKGROUND AND PURPOSE: Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available. EXPERIMENTAL APPROACH: A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability. KEY RESULTS: All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent. CONCLUSIONS AND IMPLICATIONS: It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity.

Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells.

BACKGROUND: Activation of the enzyme telomerase, which has been associated with cellular immortality, may constitute a key step in the development of human cancer. Telomerase is repressed in most normal human somatic cells. This study was conducted, using a genetic complementation approach, with the aim of identifying and mapping the genes responsible for repressing telomerase and, simultaneously, to establish the effect of experimentally induced telomerase repression on human tumor cell growth. METHODS: Individual human chromosomes isolated from normal diploid cells and tagged with bacterial antibiotic resistance genes (for later selection) were introduced into cells of the human breast carcinoma cell line 21NT by means of microcell transfer. Selected hybrid clones were screened for telomerase activity by use of the polymerase chain reaction-based telomere repeat amplification protocol (TRAP) assay, and the proliferative fate of the hybrid clones was determined. Regions of the introduced chromosomes associated with telomerase repression were mapped using segregant hybrids and a deletion analysis that employed microsatellite DNA markers. RESULTS: Strong repression of telomerase was observed following transfer of human chromosome 3 into 21NT cells but not after transfer of chromosomes 8, 12, or 20. The vast majority of hybrid clones with repressed telomerase entered permanent growth arrest after 10-18 population doublings. Deletion analysis of nonrepressed segregant monochromosome 3 hybrids indicated two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located. CONCLUSIONS: Telomerase in human breast cancer cells is efficiently repressed by a gene or genes on normal human chromosome 3p, and this repression is associated with permanent growth arrest of the tumor cells.

Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line.

BACKGROUND: High-risk human papillomavirus (HPV) types play a major role in the development of cervical cancer in vivo and can induce immortalization of primary human keratinocytes in vitro. Activation of the telomere-lengthening enzyme telomerase constitutes a key event in both processes. Because losses of alleles from chromosome 6 and increased telomerase activity have been observed in high-grade premalignant cervical lesions, we analyzed whether human chromosome 6 harbors a putative telomerase repressor locus that may be involved in HPV-mediated immortalization. METHODS: Microcell-mediated chromosome transfer was used to introduce chromosomes 6 and 11 to the in vitro generated HPV type 16 (HPV16)-immortalized keratinocyte cell line FK16A and to the in vivo derived HPV16-containing cervical cancer cell line SIHA: Hybrid clones were analyzed for growth characteristics, telomerase activity, human telomerase reverse transcriptase (hTERT) and HPV16 E6 expression, and telomere length. FK16A hybrid clones were also transduced with an hTERT-containing retrovirus to examine the effect of ectopic hTERT expression on growth. Statistical tests were two-sided. RESULTS: Introduction of human chromosome 6 but not of chromosome 11 to both cell lines yielded hybrid cells that demonstrated crisis-like features (i.e., enlarged and flattened morphology, vacuolation, and multinucleation) and underwent growth arrest after a marked lag period. In the chromosome 6 hybrid clones analyzed, telomerase activity and hTERT messenger RNA (mRNA) expression were statistically significantly reduced compared with those in the chromosome 11 hybrid clones (for telomerase activity, P =.004 for the FK16A hybrids and P =.039 for the SiHa hybrids; for hTERT mRNA expression, P =.003 for the FK16A hybrids). The observed growth arrest was associated with telomeric shortening. Ectopic expression of hTERT in FK16A cells could prevent the telomeric shortening-based growth arrest induced by chromosome 6. CONCLUSIONS: Chromosome 6 may harbor a repressor of hTERT transcription, the loss of which may be involved in HPV-mediated immortalization.

Regulation of human telomerase activity: repression by normal chromosome 3 abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity.

Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even in cells permanently expressing a transfected hTERT cDNA. However, chromosome 3 transfer did not affect the expression of green fluorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether chromosome 3 transfer affected c-Myc activity. An at least 30-fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by complex mechanisms involving regulatory elements distant from the 5' flanking region, and that the putative hTERT repressor on chromosome 3 does not regulate the expression of hTERT through c-Myc or one of its coregulators.

Human chromosome 21 determines growth factor dependence in human/mouse B-cell hybridomas.

Interleukin 6 (IL-6) serves as a growth factor for mouse plasmacytomas. As a model for IL-6-mediated growth of plasmacytomas, we study IL-6-dependent B-cell hybridomas, which can be generated through fusion of B lymphocytes with a plasmacytoma cell line, e.g., SP2/0. In the present report, we have investigated the peculiar behavior of B-cell hybridomas with respect to IL-6 dependence. We demonstrate that although newly generated hybridomas are IL-6 dependent, many hybridomas lose this dependency at frequencies as high as 50%, shortly after fusion. We speculated that the loss of IL-6-dependent growth is due to the well-known chromosomal instability of B-cell hybridomas. Consequently, loss of IL-6 dependence is the result of loss of a specific chromosome(s). This model implies the existence of an "IL-6 dependency" gene, the loss of which makes hybridomas capable of proliferating in the absence of IL-6. Because SP2/0 is IL-6 independent, the IL-6-dependent phenotype of B-cell hybridomas, and hence the IL-6 dependency gene, must be derived from the B lymphocyte. We have tested this model by generating human/mouse B-cell hybridomas through fusion of human B lymphocytes with SP2/0. We then analyzed the human chromosome content of 10 IL-6-dependent and 14 IL-6-independent subclones. From that analysis we concluded that the presence of human chromosome 21 correlated with IL-6 dependence. This correlation was confirmed by microcell fusion experiments in which a single copy of chromosome 21 was introduced into IL-6-independent hybridomas, resulting in reconstitution of the IL-6-dependent phenotype. We therefore conclude that chromosome 21 carries an IL-6 dependency gene.

Human chromosome 16 suppresses metastasis but not tumorigenesis in rat prostatic tumor cells.

Genomic aberrations at the chromosome 16q arm are one of the most consistent abnormalities observed by loss of heterozygosity and comparative genomic hybridization analyses in human prostate cancer, suggesting that there are tumor suppressor or metastasis suppressor genes encoded by this chromosomal region. To functionally identify such suppressor genes, we have conducted microcell-mediated chromosome transfer to introduce human chromosome 16 into the highly metastatic Dunning rat prostatic cancer cell line, AT6.1. The metastatic ability of the resultant microcell hybrid clones was then tested in a standard spontaneous metastasis assay using SCID mice. When the microcell-mediated chromosome transfer hybrid cells containing whole human chromosome 16 were injected, the number of metastatic lesions in the lung was significantly reduced as much as 99% on average. Therefore, chromosome 16 has a strong activity to suppress the metastatic ability of AT6.1 cells while it did not affect the tumorigenesis and tumor growth rate. A PCR analysis of various microcell hybrid clones with sequence-tagged site markers indicates that the metastasis suppressor activity is located in the q24.2 region of chromosome 16. Our results are consistent with the previous finding that the region of human chromosome 16q has frequent loss of heterozygosity in prostate cancer patients and suggest that there is a metastasis suppressor gene in this region that may play an important role in the progression of prostate cancer.

Functional evidence of novel tumor suppressor genes for cutaneous malignant melanoma.

Losses of heterozygosity involving chromosomes 9 and 10 are frequent events in the development and progression of cutaneous malignant melanoma. To investigate whether specifically deleted chromosomal regions encode tumor suppressor genes (TSGs), we introduced normal chromosome 10 into the tumorigenic human metastatic melanoma cell line UACC-903 by microcell fusion. In addition, two chromosome 9 derivatives that were microdeleted in the region of the p16INK4A/p15INK4B locus were transferred to determine whether an additional melanoma TSG or TSGs reside on chromosome 9p, as indicated by previous melanoma allele loss studies. In comparison to parental cells, microcell hybrids generated with chromosomes 9 (microdeleted) and 10 displayed reduced anchorage-independent growth in soft agar and markedly reduced tumorigenicity in athymic (nu/nu) mice. These data define a TSG or TSGs that function independently of p15/p16 on chromosome 9 and provide evidence for a TSG (or TSGs) on chromosome 10 that may be important in melanoma development.

Telomere length abnormalities in mammalian radiosensitive cells.

Telomere lengths in radiosensitive murine lymphoma cells L5178Y-S and parental radioresistant L5178Y cells were measured by quantitative fluorescence in situ hybridization. Results revealed a 7-fold reduction in telomere length in radiosensitive cells (7 kb) in comparison with radioresistant cells (48 kb). Therefore, it was reasoned that telomere length might be used as a marker for chromosomal radiosensitivity. In agreement with this hypothesis, a significant inverse correlation between telomere length and chromosomal radiosensitivity was observed in lymphocytes from 24 breast cancer patients and 5 normal individuals. In contrast, no chromosomal radiosensitivity was observed in mouse cell lines that showed shortened telomeres, possibly reflecting differences in radiation responses between primary cells and established cell lines. Telomere length abnormalities observed in radiosensitive cells suggest that these two phenotypes may be linked.

Genetic and functional analyses exclude mortality factor 4 (MORF4) as a keratinocyte senescence gene.

Approximately 50% of immortal human keratinocyte lines show loss of heterozygosity of chromosome region 4q33-q34, and the reintroduction of chromosome 4 into one such line, BICR 6, causes proliferation arrest and features of replicative senescence. Recently, a candidate gene, mortality factor 4 (MORF4), was identified in this region and sequenced in 21 immortal keratinocyte lines. There were no mutations or deletions, and two of the seven lines that showed loss of heterozygosity at 4q33-q34 were heterozygous for MORF4 itself. Furthermore, the transfer of a chromosomal segment containing the entire MORF4 gene did not mimic the senescence effect of chromosome 4 in BICR 6. These results suggest that the inactivation of MORF4 is not required for human keratinocyte immortality.

A telomere-independent senescence mechanism is the sole barrier to Syrian hamster cell immortalization.

Reactivation of telomerase and stabilization of telomeres occur simultaneously during human cell immortalization in vitro and the vast majority of human cancers possess high levels of telomerase activity. Telomerase repression in human somatic cells may therefore have evolved as a powerful resistance mechanism against immortalization, clonal evolution and malignant progression. The comparative ease with which rodent cells immortalize in vitro suggests that they have less stringent controls over replicative senescence than human cells. Here, we report that Syrian hamster dermal fibroblasts possess substantial levels of telomerase activity throughout their culture life-span, even after growth arrest in senescence. In our studies, telomerase was also detected in uncultured newborn hamster skin, in several adult tissues, and in cultured fibroblasts induced to enter the post-mitotic state irreversibly by serum withdrawal. Transfection of near-senescent dermal fibroblasts with a selectable plasmid vector expressing the SV40 T-antigen gene resulted in high-frequency single-step immortalization without the crisis typically observed during the immortalization of human cells. Collectively, these data provide an explanation for the increased susceptibility of rodent cells to immortalization (and malignant transformation) compared with their human equivalents, and provide evidence for a novel, growth factor-sensitive, mammalian senescence mechanism unrelated to telomere maintenance.