Kisspeptin treatment induces gonadotropic responses and rescues ovulation in a subset of preclinical models and women with polycystic ovary syndrome.

STUDY QUESTION: Can kisspeptin treatment induce gonadotrophin responses and ovulation in preclinical models and anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Kisspeptin administration in some anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. WHAT IS KNOWN ALREADY: PCOS is a prevalent, heterogeneous endocrine disorder, characterized by ovulatory dysfunction, hyperandrogenism and deregulated gonadotrophin secretion, in need of improved therapeutic options. Kisspeptins (encoded by Kiss1) are master regulators of the reproductive axis, acting mainly at GnRH neurons, with kisspeptins being an essential drive for gonadotrophin-driven ovarian follicular maturation and ovulation. Altered Kiss1 expression has been found in rodent models of PCOS, although the eventual pathophysiological role of kisspeptins in PCOS remains unknown. STUDY DESIGN, SIZE, DURATION: Gonadotrophin and ovarian/ovulatory responses to kisspeptin-54 (KP-54) were evaluated in three preclinical models of PCOS, generated by androgen exposures at different developmental windows, and a pilot exploratory cohort of anovulatory women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three models of PCOS were generated by exposure of female rats to androgens at different periods of development: PNA (prenatal androgenization; N = 20), NeNA (neonatal androgenization; N = 20) and PWA (post-weaning androgenization; N = 20). At adulthood (postnatal day 100), rats were subjected to daily treatments with a bolus of KP-54 (100 µg/kg, s.c.) or vehicle for 11 days (N = 10 per model and treatment). On Days 1, 4, 7 and 11, LH and FSH responses were assessed at different time-points within 4 h after KP-54 injection, while ovarian responses, in terms of follicular maturation and ovulation, were measured at the end of the treatment. In addition, hormonal (gonadotrophin, estrogen and inhibin B) and ovulatory responses to repeated KP-54 administration, at doses of 6.4-12.8 nmol/kg, s.c. bd for 21 days, were evaluated in a pilot cohort of anovulatory women (N = 12) diagnosed with PCOS, according to the Rotterdam criteria. MAIN RESULTS AND THE ROLE OF CHANCE: Deregulated reproductive indices were detected in all PCOS models: PNA, NeNA and PWA. Yet, anovulation was observed only in NeNA and PWA rats. However, while anovulatory NeNA rats displayed significant LH and FSH responses to KP-54 (P < 0.05), which rescued ovulation, PWA rats showed blunted LH secretion after repeated KP-54 injection and failed to ovulate. In women with PCOS, KP-54 resulted in a small rise in LH (P < 0.05), with an equivalent elevation in serum estradiol levels (P < 0.05). Two women showed growth of a dominant follicle with subsequent ovulation, one woman displayed follicle growth but not ovulation and desensitization was observed in another patient. No follicular response was detected in the other women. LIMITATIONS, REASONS FOR CAUTION: While three different preclinical PCOS models were used in order to capture the heterogeneity of clinical presentations of the syndrome, it must be noted that rat models recapitulate many but not all the features of this condition. Additionally, our pilot study was intended as proof of principle, and the number of participants is low, but the convergent findings in preclinical and clinical studies reinforce the validity of our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Our first-in-rodent and -human studies demonstrate that KP-54 administration in anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. As our rat models likely reflect the diversity of PCOS phenotypes, our results argue for the need of personalized management of anovulatory dysfunction in women with PCOS, some of whom may benefit from kisspeptin-based treatments. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research agreements between Ferring Research Institute and the Universities of Cordoba and Edinburgh. K.S. was supported by the Wellcome Trust Scottish Translational Medicine and Therapeutics Initiative (STMTI). Some of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. M.T.-S. is a member of CIBER Fisiopatología de la Obesidad y Nutrición, which is an initiative of Instituto de Salud Carlos III. Dr Mannaerts is an employee of Ferring International PharmaScience Center (Copenhagen, Denmark), and Drs Qi, van Duin and Kohout are employees of the Ferring Research Institute (San Diego, USA). Dr Anderson and Dr Tena-Sempere were recipients of a grant support from the Ferring Research Institute, and Dr Anderson has undertaken consultancy work and received speaker fees outside this study from Merck, IBSA, Roche Diagnostics, NeRRe Therapeutics and Sojournix Inc. Dr Skorupskaite was supported by the Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The other authors have no competing interest.

RPL5 on 1p22.1 is recurrently deleted in multiple myeloma and its expression is linked to bortezomib response.

Chromosomal region 1p22 is deleted in ⩾20% of multiple myeloma (MM) patients, suggesting the presence of an unidentified tumor suppressor. Using high-resolution genomic profiling, we delimit a 58 kb minimal deleted region (MDR) on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Low mRNA expression of EVI5 and RPL5 was associated with worse survival in diagnostic cases. Patients with 1p22 deletion had lower mRNA expression of EVI5 and RPL5, however, 1p22 deletion status is a bad predictor of RPL5 expression in some cases, suggesting that other mechanisms downregulate RPL5 expression. Interestingly, RPL5 but not EVI5 mRNA levels were significantly lower in relapsed patients responding to bortezomib and; both in newly diagnosed and relapsed patients, bortezomib treatment could overcome their bad prognosis by raising their progression-free survival to equal that of patients with high RPL5 expression. In conclusion, our genetic data restrict the MDR on 1p22 to EVI5 and RPL5 and although the role of these genes in promoting MM progression remains to be determined, we identify RPL5 mRNA expression as a biomarker for initial response to bortezomib in relapsed patients and subsequent survival benefit after long-term treatment in newly diagnosed and relapsed patients.

Pharmacogenomics and chemical library screens reveal a novel SCFSKP2 inhibitor that overcomes Bortezomib resistance in multiple myeloma.

While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1-Cullin-1-Skp2 (SCFSkp2) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCFSkp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138+ cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138+ cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCFSkp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCFSkp2 inhibitors to treat BTZ resistant disease.

Loss of CYLD expression unleashes Wnt signaling in multiple myeloma and is associated with aggressive disease.

Deletion or mutation of the gene encoding the deubiquitinating enzyme CYLD is a common genomic aberration in multiple myeloma (MM). However, the functional consequence of CYLD loss and the mechanism underlying its putative role as a tumor suppressor gene in the pathogenesis of MM has not been established. Here, we show that CYLD expression is highly variable in myeloma cell lines and primary MMs and that low CYLD expression is associated with disease progression from monoclonal gammopathy of undetermined significance to MM, and with poor overall and progression free-survival of MM patients. Functional assays revealed that CYLD represses MM cell proliferation and survival. Furthermore, CYLD acts as a negative regulator of NF-κB and Wnt/ß-catenin signaling and loss of CYLD sensitizes MM cells to NF-κB-stimuli and Wnt ligands. Interestingly, in primary MMs, low CYLD expression strongly correlated with a proliferative and Wnt signaling-gene expression signature, but not with an NFκB target gene signature. Altogether, our findings identify CYLD as a negative regulator of NF-κB and Wnt/ß-catenin signaling in MM and indicate that loss of CYLD enhances MM aggressiveness through Wnt pathway activation. Thus, targeting the Wnt pathway could be a promising therapeutic strategy in MM with loss of CYLD activity.

(18)F-fluoride-PET for dynamic in vivo monitoring of bone formation in multiple myeloma.

BACKGROUND: Bone disease in multiple myeloma is characterized by reduced bone formation. The gold standard of bone formation is the mineral apposition rate (MAR), an invasive technique reflecting bone formation at a single site. We compared (18)F-fluoride-PET with the MAR in myeloma patients. METHODS: Bone formation was measured before and after bortezomib treatment by determination of the MAR in iliac bone marrow biopsies and the measurement of (18)F-uptake. RESULTS: The inter- and intra-individual variations in (18)F-uptake (SUVA50%) were pronounced as 33.50 (range 4.42 to 37.92) and 27.18 (range 4.00 to 31.18), respectively. A significant correlation between the MAR and (18)F-uptake was found (r = 0.80, p = 0.017). There was a heterogeneous response after treatment varying from -2.20 to 4.53. CONCLUSIONS: Iliac (18)F-uptake was associated with the local MAR in myeloma patients. Furthermore, (18)F-fluoride-PET demonstrated the heterogeneity of in vivo bone formation, enabling monitoring during treatment.

Gene expression profile alone is inadequate in predicting complete response in multiple myeloma.

With advent of several treatment options in multiple myeloma (MM), a selection of effective regimen has become an important issue. Use of gene expression profile (GEP) is considered an important tool in predicting outcome; however, it is unclear whether such genomic analysis alone can adequately predict therapeutic response. We evaluated the ability of GEP to predict complete response (CR) in MM. GEP from pretreatment MM cells from 136 uniformly treated MM patients with response data on an IFM, France led study were analyzed. To evaluate variability in predictive power due to microarray platform or treatment types, additional data sets from three different studies (n=511) were analyzed using same methods. We used several machine learning methods to derive a prediction model using training and test subsets of the original four data sets. Among all methods employed for GEP-based CR predictive capability, we got accuracy range of 56-78% in test data sets and no significant difference with regard to GEP platforms, treatment regimens or in newly diagnosed or relapsed patients. Importantly, permuted P-value showed no statistically significant CR predictive information in GEP data. This analysis suggests that GEP-based signature has limited power to predict CR in MM, highlighting the need to develop comprehensive predictive model using integrated genomics approach.

A gene expression signature for high-risk multiple myeloma.

There is a strong need to better predict the survival of patients with newly diagnosed multiple myeloma (MM). As gene expression profiles (GEPs) reflect the biology of MM in individual patients, we built a prognostic signature based on GEPs. GEPs obtained from newly diagnosed MM patients included in the HOVON65/GMMG-HD4 trial (n=290) were used as training data. Using this set, a prognostic signature of 92 genes (EMC-92-gene signature) was generated by supervised principal component analysis combined with simulated annealing. Performance of the EMC-92-gene signature was confirmed in independent validation sets of newly diagnosed (total therapy (TT)2, n=351; TT3, n=142; MRC-IX, n=247) and relapsed patients (APEX, n=264). In all the sets, patients defined as high-risk by the EMC-92-gene signature show a clearly reduced overall survival (OS) with a hazard ratio (HR) of 3.40 (95% confidence interval (CI): 2.19-5.29) for the TT2 study, 5.23 (95% CI: 2.46-11.13) for the TT3 study, 2.38 (95% CI: 1.65-3.43) for the MRC-IX study and 3.01 (95% CI: 2.06-4.39) for the APEX study (P<0.0001 in all studies). In multivariate analyses this signature was proven to be independent of the currently used prognostic factors. The EMC-92-gene signature is better or comparable to previously published signatures. This signature contributes to risk assessment in clinical trials and could provide a tool for treatment choices in high-risk MM patients.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene-diene rubber using gas chromatography and mass spectrometry II. Addition and combination reactions.

The dicumyl-peroxide-initiated addition and combination reactions of mixtures of alkanes (n-octane, n-decane) and alkenes [5,6-dihydrodicyclopentadiene (DCPDH), 5-ethylidene-2-norbornane (ENBH) and 5-vinylidene-2-norbornane (VNBH)] were studied to mimic the peroxide cross-linking reactions of terpolymerised ethylene, propylene and a diene monomer (EPDM). The reaction products of the mixtures were separated by both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GCxGC). The separated compounds were identified from their mass spectra and their GC and GCxGC elution pattern. Quantification of the various alkyl/alkyl, alkyl/allyl and allyl/allyl combination products shows that allylic-radicals comprise approximately 60% of the substrate radicals formed. The total concentration of the products formed by combination is found to be independent of the concentration and the type of alkene. The total concentration of the products formed by addition to the alkene increases with increasing concentration of alkene. In addition, the total concentration of the formed addition products depends strongly on the type of the alkene used, viz. VNBH>ENBH approximately DCPDH, which is a consequence of differences in steric hindrance of the unsaturation. The peroxide curing efficiency, defined as the number of moles of cross-linked products formed per mol of peroxide, is 173% using 9% (w/w) 5-vinylidene-2-norbornane (VNBH). This indicates that the addition reaction is recurrent. All these findings are consistent with experimental studies on peroxide curing of EPDM rubber. In addition, the present results provide more-detailed structural information, increasing the understanding of the mechanism of peroxide curing of EPDM. The described approach to use low-molecular-weight model compounds followed by GC-mass spectrometry (MS) and GCxGC-MS analysis is proven to be a very powerful tool to study the cross-linking of EPDM.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene (-diene) rubber using gas chromatography and mass spectrometry I. Combination reactions of alkanes.

The combination reaction of linear and branched alkanes, initiated by dicumylperoxide, has been studied as a model for the combination cross-linking reaction of peroxide-cured terpolymerised ethylene, propylene and diene monomer. Both gas chromatography-mass spectrometry (GC-MS) and comprehensive two-dimensional GC-MS (GCxGC-MS) analyses have been employed to analyse the isomeric reaction products. The identification of these products based on their MS fragmentation patterns is quite complex, due to the high tendency of random rearrangements. Careful elucidation of the high-mass ions at optimised ionisation energy (55eV) has resulted in proposed structures for the different isomeric reaction products. The structure assignment by MS is in agreement with the GCxGC elution pattern and with the result of a theoretical model to predict the boiling points and, thus, the GC retention times. In addition, a model that provided a direct correlation between chemical structure and retention times was developed and this was found to provide a useful fit. Quantification of the identified reaction products by GC separation and flame ionization detection allows classification according to the hydrogen abstraction sites for the alkanes by dicumylperoxide. The selectivity for hydrogen abstraction generally follows the expected order, but a higher reactivity was observed for the methylene group next to a primary methyl group, while a reduced reactivity of the methylene group next to ethyl and to methyl groups was observed. The used approach proved to be a very powerful tool to enhance our understanding of the mechanism of peroxide cross-linking of (branched) alkanes.

Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels.

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22-q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin-dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto-oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barrett's dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high-resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.

High-resolution array comparative genomic hybridization of chromosome 8q: evaluation of putative progression markers for gastroesophageal junction adenocarcinomas.

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124-125 Mb (8q24.13), at 127-128 Mb (8q24.21), and at 141-142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas.

Genomics in target and drug discovery.

Genomics-based discovery of novel therapeutic drug targets requires the design of well-controlled biological or pharmacological experiments with experimental questions and hypotheses that relate to the therapeutic area of interest. This will aid the validation level of differentially expressed genes and hence facilitate the de-selection of the genes that are identified in microarray experiments. We here provide an example of how this approach is followed in the manipulation of human macrophage foam cells towards the discovery of novel drug targets for treatment of atherosclerosis.

Cloning and characterization of the cyclic guanosine monophosphate-inhibited phosphodiesterase PDE3A expressed in mouse oocyte.

In the preovulatory follicle, oocyte meiotic resumption occurs soon after the LH surge and is associated with a decrease in cAMP. Inhibition of cAMP degradation blocks germinal vesicle breakdown as well as activation of meiotic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacological analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compartment. Here we have investigated the structure and properties of the PDE form expressed in mouse oocytes. Polymerase chain reactions using a mouse oocyte cDNA library as a template, and primers based on the conserved sequence of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent ligation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this PDE, the cDNAs encoding the full-length PDE3A and NH(2)-truncation forms Delta 1 (Delta346aa) and Delta 2 (Delta608aa) were expressed in mouse Leydig tumor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly in the soluble fraction. The Michaelis constant values for hydrolysis of cAMP of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact full-length PDE3A or oocyte PDE (0.2-0.5 microM). More importantly, there was good correlation between the rank of potency of selective and nonselective compounds in inhibiting recombinant PDE3A or PDE activity derived from cumulus-oocyte complexes and in blocking resumption of meiosis. These data provide evidence that the PDE expressed in the oocyte is a soluble form of PDE3A and that activity of this enzyme is involved in the control of resumption of meiosis.

[Human papillomavirus and the development of cervical cancer: concept of carcinogenesis]. / Humaan papillomavirus en het ontstaan van baarmoederhalskanker: concept van carcinogenese.

Infection with high risk human papillomavirus (hrHPV) plays a central aetiological role in cervical cancer. Still, cervical carcinogenesis is a multistep process which requires other events in addition to hrHPV infection. Recent data have resulted in the following concept of cervical carcinogenesis: hrHPV infects normal squamous epithelium. In most cases this will not lead to a lesion or at worst give rise to a regressing low grade cervical intraepithelial neoplasia (CIN). Both phenomena involve viral clearance. Only persistent hrHPV infections will lead to a high grade CIN lesion, a subset of which may undergo malignant transformation. At the transition of CIN 2 to CIN 3 deregulated expression of the viral oncogenes E6 and E7 takes place, resulting in genetic instability. Subsequently, activation of the telomere-lengthening enzyme, telomerase occurs, at the result of which cells obtain an infinite replication capacity. Ultimately, successive allele losses occur at different chromosomal locations which, followed by a clonal outgrowth result in an invasive carcinoma.

The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7.

Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.

Analysis of human papillomavirus type 16 E6 variants in relation to p53 codon 72 polymorphism genotypes in cervical carcinogenesis.

This study aimed to assess the role of specific human papillomavirus type 16 (HPV-16) variants, in combination with p53 codon 72 polymorphism genotypes, in cervical carcinogenesis. An initial sequence analysis of HPV-16 long control, E6 and E7 regions of 53 well-defined cervical samples containing HPV-16 revealed that a T to G transition at nucleotide position 350 within the E6 open reading frame was the most common variation, the frequency of which seemed to decrease with increasing severity of the lesion. Therefore, a total of 246 cervical samples of residents of The Netherlands was specifically analysed for HPV-16 350G/T variants and/or p53 codon 72 genotypes. These comprised HPV-negative normal cervical scrapes (n=40), normal cervical scrapes containing HPV-16 (n=46), scrapes containing HPV-16 from women with abnormal cervical cytology participating in a non-intervention follow-up study without (n=38) and with (n=51) a histologically proven cervical intraepithelial neoplasia (CIN) III lesion at the end of the study, and cervical squamous cell carcinomas (n=71). Neither specific HPV-16 350G/T variants nor specific p53 genotypes were associated with a higher risk of developing CIN III or cervical cancer. However, HPV-16 350T variants were significantly over-represented in p53 Arg homozygous women with cervical cancer. This suggests that, in p53 Arg/Arg women, infection with HPV-16 350T variants confers a higher risk of cervical cancer.

Daunorubicin-induced pathology in the developing hamster molar tooth germ in vitro.

The aim of this study was to evaluate, under organ culture conditions, the cytotoxic effects of daunorubicin on tooth development. Three-day-old maxillary hamster second molars were exposed for 24 h in vitro to 108-10-4 M daunorubicin and then evaluated biochemically and histologically. At 10-6 M daunorubicin dose-dependently decreased tooth germ dry weight, cell proliferation ([3H]thymidine uptake), and insoluble [32P] phosphate uptake (phosphorylation of macromolecules). [45Ca]calcium uptake, a marker for mineralization, was significantly affected only at the highest concentration (10-4 M) tested. Histologically, 10-6 M daunorubicin induced necrosis of the proliferating but not the differentiated protein-secreting cells. At 10-4 M, however, all cells were dead. These results indicate that daunorubicin is particularly toxic to the proliferating cells of the tooth germ. Thus, it can be postulated that children treated with daunorubicin may develop defects in the erupted teeth mainly associated with those regions that were in the proliferating stage at the onset of anticancer chemotherapy.

HPV 16 infection and progression of cervical intra-epithelial neoplasia: analysis of HLA polymorphism and HPV 16 E6 sequence variants.

High-risk human papillomavirus (HPV) infection plays an important role in cervical intra-epithelial neoplasia (CIN), but HPV infection alone is not sufficient for progression to cervical cancer. Several lines of evidence suggest that cellular immune surveillance is important in the control of HPV infection and the development of CIN. The presentation to T cells of target viral peptides in the context of HLA molecules is influenced by the genetic polymorphisms of both HPV and HLA and thereby influences the host immune response and clinical outcome of HPV infection. HLA class I and II polymorphism in susceptibility for HPV 16 infection, development and progression of CIN was analyzed in a group of 118 patients participating in a prospective study of women with initial abnormal cytology. Patients were stratified according to HPV status and course of the disease. HLA-B*44 frequency was increased in the small group of patients with a lesion that showed clinical progression during follow-up [OR = 9.0 (4.6-17.5), p = 0.007]. HLA-DRB1*07 frequency was increased among HPV 16-positive patients compared with patients who were negative for all HPV types [OR = 5.9 (3.0-11.3), p = 0.02]. Our results are consistent with the immunogenetic factors associated with disease progression being different from those associated with susceptibility to HPV 16 infection. Sequencing of the HPV 16 E6 and E7 open reading frames of a subset of these patients (n = 40) showed the frequency of HPV 16 variants to be similar to other studies. However, there was no significant correlation between variant incidence and disease progression or viral persistence and no significant correlation with any HLA allele. It appears that multiple HLA types can influence HPV 16-associated cervical dysplasia but the role of HPV 16 variants in disease progression and susceptibility in relation to HLA polymorphism remains unclear.

Telomerase activity exclusively in cervical carcinomas and a subset of cervical intraepithelial neoplasia grade III lesions: strong association with elevated messenger RNA levels of its catalytic subunit and high-risk human papillomavirus DNA.

In this study, we investigated telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression in relation to high-risk human papillomavirus (HPV) DNA presence in the spectrum of cervical premalignant lesions. Reconstruction experiments revealed that telomerase activity determined by the telomeric repeat amplification protocol assay and hTERT mRNA by reverse transcriptase-PCR could be detected in down to 100 and 1 SiHa cervical cancer cells, respectively. Telomeric repeat amplification protocol analysis on cervical tissue specimens revealed that none of the histomorphologically normal cervical samples (n = 8) and cervical intraepithelial neoplasia (CIN) grade I (n = 10) and grade II (n = 8) lesions had detectable telomerase activity. However, telomerase activity was shown in 40% of CIN grade III lesions (n = 15) and 96% of squamous cell carcinomas (n = 24). Despite the fact that hTERT mRNA was found at much higher frequencies, semiquantitative reverse transcriptase-PCR revealed that elevated hTERT mRNA levels were strongly correlated with detectable telomerase activity. Furthermore, telomerase activity and elevated hTERT mRNA levels were only detected in cases that contained high-risk HPV DNA. In contrast, low or undetectable hTERT mRNA levels were demonstrated in both high-risk HPV positive and negative cases. These data indicate that telomerase activity detectable with the assay used and concomitant elevated levels of hTERT mRNA reflect a rather late step in the CIN to squamous cell carcinoma sequence, which follows infection with high-risk HPV.