[Non-pharmacological treatment of hospital patients with sleeping problems - the nurse perspective]. / Nicht-medikamentöse Maßnahmen bei Ein- und Durchschlafproblemen von älteren Patienten im Krankenhaus ­ Qualitative Interviews mit Pflegenden.

Non-pharmacological treatment of hospital patients with sleeping problems - the nurse perspective Abstract. BACKGROUND: Elderly patients suffer from sleep disturbances during hospitalization. These patients often receive hypnotics and sedatives; despite of the known risks and although non-pharmacological treatments are available. AIM: The study investigates the experiences of nurses when using non-pharmacological treatments for elderly patients with sleeping problems. METHODS: Semi-structured interviews with 13 nurses from a general hospital were analyzed according to Mayring's qualitative content analysis. RESULTS: Nurses used a variety of non-pharmacological treatments for elderly inpatients with sleeping problems: (1) structural measures (regulation of temperature and light), (2) organizational measures (more time for conversation during the nightshift), (3) nursing measures (asking about night-time routines) and (4) household remedies. From the nurses' perspective, the more intensive contact required when applying non-pharmacological treatments can lead to higher patient satisfaction and a lower bell frequency during the night shift. Barriers result from limited time and personnel, a lack of standards and individual patient needs. CONCLUSION: Nurses know several kinds of non-pharmacological treatments to help elderly inpatients sleep better. A lack of resources as well as a lack of professional consensus about the treatment of temporary sleeping disturbances can be an obstacle to their use. A professional climate should restrict the use of drugs for sleeping problems as far as possible.

High frequency of promoter methylation of the 14-3-3 sigma and CAGE-1 genes, but lack of hypermethylation of the caveolin-1 gene, in primary adenocarcinomas and signet ring cell carcinomas of the urinary bladder.

The molecular mechanisms underlying the histogenesis of nonurothelial carcinomas of the urinary bladder are not yet clearly understood. There is a growing body of evidence that, generally, epigenetic regulation mediated by methylation of normally unmethylated CpG islands at the 5' promoter regions of genes is involved in the modification of tumorigenesis. This prompted the current study to explore the methylation status of a broad panel of various genes implicated in cell differentiation and tumor suppression in 10 adenocarcinomas and 6 signet ring cell carcinomas of the bladder. Using methylation-specific PCR, we were able to detect a high frequency of promoter methylation of the 14-3-3 sigma (100%) and CAGE-1 (80%) genes in adenocarcinomas, and in nearly all signet ring cell carcinomas. The SYK and hDAB2IP genes proved to be hypermethylated in only single cases, whereas the caveolin-1 gene failed to be hypermethylated in all cases. The present data suggest that promoter methylation of the 14-3-3 sigma and CAGE-1 genes plays a crucial role during the phenotypical morphogenesis of vesical adenocarcinomas including signet ring cell carcinomas by an epigenetic mechanism.

Detection and analysis of cancer genes amplified from bone material of a Scythian royal burial in Arzhan near Tuva, Siberia.

Molecular paleopathology has become an emerging field that helps to characterize molecular markers of past disease. Especially highly sensitive genetic techniques such as PCR are an important means of unraveling changes in ancient DNA extracted from bone tissue, teeth and mummified soft tissue. In the present study, excavated bone material from the skeleton of a Scythian sovereign, morphologically and immunohistochemically suspicious of a metastatic prostate carcinoma, was analyzed by PCR for amplifiable human gene sequences. Short sequences of the human GADD153 DNA repair gene and p53 tumor suppressor gene were detectable which revealed the absence of mutations according to the data of automatic sequencing. Using bisulfite-treated DNA from the bone, methylation-specific PCR detected hypermethylated promoter sequences of the p14ARF tumor suppressor gene. In summary, these data show that it is possible: a) to amply short human DNA stretches from 2,500-year-old bone material, b) to detect tumorigenetically important genes within this DNA, c) to detect epigenetically modified DNA in ancient bone material. The finding of hypermethylated p14ARF sequences merits attention because this may indicate an intraosseal neoplastic process and may corroborate the hypothesis of prostate cancer.

Promoter hypermethylation of the 14-3-3 sigma, SYK and CAGE-1 genes is related to the various phenotypes of urinary bladder carcinomas and associated with progression of transitional cell carcinomas.

To explore the significance of epigenetic mechanisms in urinary bladder carcinogenesis mediated by methylation of cytosine in CpG dinucleotides at 5' promoter regions, we analysed the methylation status of a broad panel of different genes in transitional cell carcinomas (TCC) and nonurothelial cancers, among which the 14-3-3 sigma, SYK and CAGE-1 genes were recognised as promising target genes. Using methylation-specific PCR, the rate of DNA hypermethylation proved to be related to the various histopathological cancer subtypes. The higher frequency of promoter methylation of the 14-3-3 sigma (57.1%) and SYK (64.3%) genes in high-grade, high-stage TCC in association with a reduced or even lacking immunohistochemical protein expression than in low-grade, low-stage TCC (28.6% and 42.9%, respectively), indicates that aberrant methylation of these genes plays an essential role in the progression of TCC. The importance of DNA hypermethylation in the conversion of TCC from a low to a high malignant potential was strongly supported by the finding that, unlike superficial low-grade TCC, advanced muscle invasive TCC showed a concurrent promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes. Squamous cell carcinomas revealed a peak incidence of hypermethylation of the 14-3-3 sigma gene (80%), and conversely, the lowest methylation frequency of the SYK gene (13.3%). Undifferentiated small cell carcinomas disclosed a promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in only a quarter each for the cases. Although a correlation between the methylation status and gene activity in squamous cell and undifferentiated small cell carcinomas was not observed, the underexpression of the SYK protein products in both cancer types and additionally of the 14-3-3 sigma protein in small cell carcinomas appeared to be related to the aggressive clinical behaviour of both these nonurothelial bladder carcinomas. The relevance of the high frequency of DNA hypermethylation of the CAGE-1 antigen in TCC and squamous cell carcinomas merits further study, particularly in relation to anticancer immunotherapy. The methylation status of the PTEN, COX-2, RUNX-3 and HIC-1 genes was found to be unaltered. In conclusion, the different patterns of aberrant methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in the various histopathological cancer types of the urinary bladder point to a role in tumor cell differentiation, resulting in the phenotypical conversion of TCC into nonurothelial carcinomas and in the progression of TCC to a more malignant potential.

Transitional cell carcinomas and nonurothelial carcinomas of the urinary bladder differ in the promoter methylation status of the caveolin-1, hDAB2IP and p53 genes, but not in the global methylation of Alu elements.

Tumor suppressor genes play a prominent role in the modification and progression of urinary bladder carcinogenesis as a result of classic genetic alterations. Little is known about the potential significance of epigenetic events, mediated by DNA hypermethylation. This prompted our investigation to explore the global Alu methylation and the promoter methylation of the novel putative tumor suppressor genes caveolin-1 and hDAB2IP, and of p53 in transitional cell carcinomas (TCC), squamous cell carcinomas and undifferentiated small cell carcinomas of the urinary bladder. Quantitative GeneScan analysis revealed that the various histopathological tumor entities showed considerable interindividual variations in the global methylation, but the overall rate did not significantly differ between the various cancer subtypes. With methylation-specific PCR, a high frequency of methylation of the promoter region of the caveolin-1 gene was detected in undifferentiated small cell carcinomas (50%) and in squamous cell carcinomas (25.9%), while TCC were found not to be methylated. By immunohistochemistry, all squamous cell carcinomas showed a strong diffuse overexpression of caveolin-1, whereas undifferentiated small cell cancers lacked any expression. High-grade, high-stage TCC disclosed a higher incidence (60%) and a substantially stronger expression than low-grade, low-stage TCC (42.9%). Our findings suggest that hypermethylation of the caveolin-1 gene and an abnormal protein expression play a crucial role in cell differentiation, and in the phenotypical conversion of TCC into nonurothelial carcinomas. Promoter methylation of the hDAB2IP gene occurred more frequently in advanced muscle invasive (72.7%) than in superficial noninvasive (50%) TCC. DNA hypermethylation of p53 was detected in a quarter of the low-grade, low-stage TCC and undifferentiated small cell carcinomas, but only sporadically in squamous cell carcinomas, and was absent in high-grade, high-stage TCC. In conclusion, aberrant methylation and abnormal protein expression of the caveolin-1-gene is involved in the formation of nonurothelial carcinomas of the urinary bladder and promoter methylation of the hDAB2IP gene in the progression of TCC from a low to a high malignant potential.

Alterations of the retinoblastoma and p16 pathway correlate with promoter methylation in malignant fibrous histiocytomas.

Recent reports indicate that the alterations in the p16 and pRb pathways can influence tumour progression and poor prognosis in several tumours. The objective of this study was to analyse p16 and pRb expression in161 patients with malignant fibrous histiocytomas (MFH). By immunohistochemistry, p16 and pRb were demonstrated in 25% and 56% of MFH, respectively. Cox regression analysis demonstrated an independent prognostic influence of both genes. Generally, the loss of p16 and pRb expression correlated with poorer prognosis. Promoter methylation of p16 was found in 16/42 of p16 negative MFH and of pRb in 2/42 of pRb-negative MFH. It can be concluded that p16 and pRb alterations play an important role in the progression of soft tissue sarcomas.

Human tissue for in vitro research as an alternative to animal experiments: a charitable "honest broker" model to fulfil ethical and legal regulations and to protect research participants.

Research with human tissue offers the possibility not only of improving preclinical pharmaceutical research and safety assessment, but also of the substitution of some animal experiments. Surgically removed human tissue is discarded after pathological evaluation. This tissue would be of enormous value for research, especially in the pharmaceutical branch, if it were readily available in an ethically and legally approved manner. But there are public concerns about the use of human tissue, especially for "commercial" purposes, such as in the pharmaceutical industry. The question is whether the ethical boundaries are sufficiently respected in the course of striving for industrial profit. To overcome this problem, a clear procedure for tissue donation, collection, supply and allocation must be established, which is guaranteed to be independent of special interests. The persisting problem seems to be the lack of an authority which asks for informed consent, coordinates tissue as well as blinded data collection, and supplies research facilities with tissue samples in a transparent manner. Therefore, a charitable, state-controlled foundation acting as an "honest broker" was initiated, to cover the ethical and legal aspects, as well as to protect the research participants in their use of human tissue as an alternative to animal experiments.

[Protestant clergymen among Hahnemann's clientele. Patient histories in letters]. / Evangelische Geistliche in Hahnemanns Patientenschaft. Krankengeschichten in Briefen.

As part of the research project, developments in the history of science and in the regional and ecclesiastic history of the late feudal petty state of Köthen-Anhalt have been assessed and numerous documents of the Nagel and Mühlenbein family histories examined that place the transcribed patient letters of the two Protestant clergymen within the context of the Hahnemann Archives. These findings complement and extend previous insights into Hahnemann's Köthen clientele, especially when it comes to the structure and milieu of the local clerical elite. Inspired by the interpretive methods of sequential textual analysis, form and content of the letters of the two clergymen and their relatives were also investigated as methodically structured lines of communication. The body of sources published here presents--embedded in the body-image (of sickness and health) prevalent at the time--the medical cultures of educated patients as well as the increasingly professionalized medical practices of Samuel Hahnemann in a flourishing urban doctor's surgery. The correspondence between the pastors Albert Wilhelm Gotthilf Nagel (1796-1835) and August Carl Ludwig Georg Mühlenbein (1797-1866), presented here in a standard edition, has been investigated at Fulda University as part of the project 'Homöopathisches Medicinieren zwischen alltäglicher Lebensführung und professioneller Praxis' ('Homeopathic medicine between everyday use and professional practice'). Of the altogether 78 transcribed documents, 53 are letters written by either of the two pastors, 16 are patient journals by Samuel Hahnemann, 9 letters by the pastors' wives and Mühlenbein's mother. The two series of letters, originally composed between 1831 and 1833 in old German cursive script, can now be used as sources for research into the history of homeopathy.

Abundant expression of spliced HDM2 in Hodgkin lymphoma cells does not interfere with p14(ARF) and p53 binding.

Recently, comparative genomic hybridization (CGH)- and fluorescence in situ hybridization (FISH)-analyses of native Hodgkin and Reed-Sternberg (H&RS) cells extracted from Hodgkin lymphoma (HL) revealed a recurrent amplification of the HDM2 locus on chromosome 12. HDM2 is known to target, inactivate and to degrade p53. Wild type (wt) p53 protein is detected in high levels in HL. Simultaneously, stabilized wt p53 and spliced hdm2 transcripts have been observed in different tumors. Therefore, we examined the expression and structure of HDM2 in HL cell lines and possible effects on components of the p53 pathway. DNA integrity and induction potential of p53 was verified by DNA sequencing and detection of potential effector proteins (p21(WAF/CIP), HDM2) using immunofluorescence, respectively. All HL cell lines show an overexpression of HDM2 protein. Furthermore, several different spliced hdm2 transcripts (mdm-sv) including five new variants lacking a functional p53 binding site were characterized. If expressed, corresponding proteins were shown to be not restricted to the nucleus. Co-localization of the potential binding partners HDM2/p14(ARF) and HDM2/p53 was found in HL cell lines. We suggest that HDM2-sv have no significant disturbing influence on the interaction of these proteins.

Alteration of the MDM2-p73-P14ARF pathway related to tumour progression during urinary bladder carcinogenesis.

Transitional cell carcinomas (TCC) of the urinary bladder develop by a multistep process characterized by various stages of transformation differing in their grade of malignancy and biological behaviour. Since the prospective clinical outcome cannot be reliably predicted on histopathological grounds, we analysed the mRNA expression of the MDM2-p73-P14ARF tumour surveillance pathway in an attempt to detect alterations of gene activity, allowing a better understanding of the mechanisms responsible for conversion of low to high malignant TCC. Expression of the mRNA was determined in 71 TCC of various grades and stages using the real-time quantitative reverse transcription-polymerase chain reaction. The MDM2-p73-P14ARF pathway was dominated by the MDM2 gene, the mRNA expression of which proved to be significantly (5-fold) lower in advanced high-grade, high-stage than in superficial low-grade, low-stage TCC. Conversely, the expression of p73 mRNA increased with increasing tumour grades and stages, while the activity of the P14ARF gene was not substantially altered during early and late phases of urothelial carcinogenesis. Analysing the expression of spliced variants of MDM2 mRNA, we found a heterogeneous pattern including a novel splicing transcript coding for an abnormal protein. Promoter hypermethylation of P14ARF occurred in 10% of the TCC with an under-expression of mRNA. An analysis of the effects of lifestyle and occupational bladder cancer risk factors revealed that TCC of smokers showed a 2-fold elevated expression of MDM2 mRNA and an approximately 2-fold lower expression of P14ARF mRNA, whereas the activity of the p73 gene was unchanged. Heavy coffee consumption was associated with a 2-fold decreased expression level of P14ARF mRNA. Exposure to certain occupational hazards (plastic products, paints and lacquer, polycyclic hydrocarbons, chemical solvents) was observed to modulate the activity of the genes analysed. Our findings suggest that an alteration in the MDM2-p73-P14ARF pathway is involved in the progression of bladder cancer to a more malignant and aggressive form.

Alteration of the vascular endothelial growth factor and angiopoietins-1 and -2 pathways in transitional cell carcinomas of the urinary bladder associated with tumor progression.

Neoangiogenesis is assumed to play an important role in the progression, metastasis and prognosis of a wide variety of tumors. To get insights into the molecular-genetic pathways and the biological role of angiogenesis in urothelial carcinogenesis, we analyzed comparatively the expression of the mRNA of the vascular endothelial growth factor (VEGF) and of the angiopoietins-1 and -2 (Ang-1 and Ang-2) in 71 transitional cell carcinomas (TCC) of the urinary bladder in relation to the tumor grades and stages, and referring to epidemiological risk factors. Using real-time quantitative reverse transcription-polymerase chain reaction, low-stage superficial TCC expressed VEGF and Ang-2 mRNA at a significantly higher level than high-stage muscle invasive carcinomas, and low-grade TCC at an insignificantly higher level than high-grade tumors. The activity of both angiogenic factors was found to be significantly correlated. Conversely, Ang-1 mRNA was expressed at a 3-fold significantly lower level in low-grade, low-stage compared to high-grade, high-stage TCC. A significantly 3- and 2-fold respectively, drop of the VEGF and Ang-2 mRNA expression in conjunction with a 2-fold significantly higher expression of Ang-1 mRNA in the group of grade 2 TCC when infiltrating the muscle layer may represent a crucial event during urothelial carcinogenesis, and possibly indicates an important step in promoting the conversion of bladder cancer from a low to a high malignancy in this subset of carcinomas. By immunhistochemistry, high-grade, high-stage carcinomas less frequently displayed areas with a strong reactivity for the VEGF protein ('hot spots") than low-grade, low-stage TCC, paralleling the expression of the mRNA. The expression patterns observed are compatible with a reduced vascular destabilization and decreased formation of new blood vessels in advanced TCC, suggesting a balance between vessel regression and vascular growth, with a less pronounced vascular remodeling during late phases of urothelial carcinogenesis. Analyzing the effect of life-style bladder cancer risk factors, habitual smoking and coffee consumption was not observed to substantially alter the expression of the angiogenic mediators, except for weakly elevated levels of VEGF and Ang-2 mRNA in TCC of strong smokers and a borderline significantly decreased VEGF mRNA expression associated with heavy coffee consumption. Certain hazardous occupational exposures (polycyclic hydrocarbons, paints and lacquer, stone dust) may play a role in modulating tumor angiogenesis. The current data indicate that the signaling molecular-genetic pathways underlying vascular remodeling are involved in the progression of urinary bladder cancer to a more malignant and aggressive behaviour.

Altered expression and new mutations in DNA mismatch repair genes MLH1 and MSH2 in melanoma brain metastases.

Brain metastases, including those of malignant melanoma (known for its high genomic instability), are the most common intracranial tumors. The main objective of this study was to investigate expression and mutation in the DNA mismatch repair system in melanoma brain metastases. Expression of MLH1, MSH2, PMS1 and PMS2 was investigated immunohistochemically in 31 melanoma metastatic tumors. Mutational analysis of MLH1 and MSH2 was performed in 17 melanoma brain metastases. Loss of MLH1 and MSH2 expression was found in 10/31 and 12/31 tumors. PMS1 (27/31) and PMS2 (28/31) expression was preserved in the majority of lesions. Potential missense mutation was found in MSH2 (exon 13) in 2/17 melanomas. Mutation in the intron sequence between exon 14 and 15 of MLH1 (exon 15) was observed in 4/17 cases. Our results indicate that the two major DNA mismatch repair genes, MLH1 and MSH2, are more frequently affected by alterations in the DNA mismatch repair system than the helper genes PMS1 and PMS2. The presence of mutations of MSH2 and MLH1 in melanoma brain metastases, which has not been found in primary melanomas, indicates the high genomic instability of melanoma brain metastases.

Altered mRNA expression of the Rb and p16 tumor suppressor genes and of CDK4 in transitional cell carcinomas of the urinary bladder associated with tumor progression.

Based on the concept that tumor suppressor genes are involved in the pathogenesis of urinary bladder carcinogenesis, we analysed the mRNA expression of the retinoblastoma (Rb) and p16 (CDKN2, INK4A, MTS1) genes as well as of the proto-oncogene cyclin D-dependent kinase 4 (CDK4) in 71 transitional cell carcinomas (TCC) of the urinary bladder in relation to the tumor grades and stages, and with reference to certain lifestyle and occupational risk factors. Using real-time quantitative reverse transcription-polymerase chain reaction, high-stage muscle invasive TCC expressed the Rb, p16 and CDK4 mRNA at lower levels than low-stage superficial cancers, indicating down-regulation to be linked with tumor progression. The drop of the expression in the group of grade 2 TCC when invading the muscle layer compared to grade 2 carcinomas with a superficial pattern of growth is considered to represent a key event in promoting urothelial carcinogenesis in this subset of carcinomas. The protein expression of the Rb gene evaluated by immunohistochemistry proved to be closely related to the tumor grades and stages as well as to the mRNA expression, high-grade and high-stage TCC disclosing a lower rate of positive immunoreactivity than low-grade and low-stage carcinomas. The p16 protein product was expressed at a lower level in grade 3 than in grade 1 TCC, but there was no correlation with the tumor stages or the mRNA expression. TCC with loss of heterozygosity (LOH) at the INK4A region showed a decreased expression of p16 mRNA compared to those without an allelic loss. Tobacco smoke was not identified to substantially modulate the Rb/p16/CDK4 pathways, except for a ten-fold elevated mRNA expression of the p16 gene in TCC of light compared to heavy smokers. Heavy coffee consumption was associated with a reduced expression of CDK4 mRNA. Among occupational exposures, TCC of patients in contact with stone dust, paints and lacquer, plastics, wood and wood preservers and chemical solvents and adhesives displayed altered partly elevated, partly reduced levels of Rb, p16 and CDK4 mRNA compared to non-exposed subjects. Although the underlying molecular-genetic pathways are not yet fully understood, the current results suggest functional reduction of the tumor suppressor genes Rb and p16 to be associated with progression of bladder cancer to a more malignant and aggressive behaviour.

Detection of MDM2 alterations in cultured human hepatocytes treated with 17beta-estradiol or 17alpha-ethinylestradiol.

BACKGROUND: Preneoplastic and neoplastic lesions of the liver are suspected to arise as a result of estrogen treatment. Here we present the first report on the modulational effects of the steroids 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) on oncogene MDM2 in human hepatocytes. MATERIALS AND METHODS: Collagen-embedded cultures of hepatocytes stimulated with different E2/EE2 concentrations were analyzed by immunocytochemistry, RT-PCR and sequencing for MDM2 protein/mRNA expression, MDM2 mRNA splicing and MDM2 gene mutation. RESULTS: The hepatocytes responded to stimulation with steroid E2/EE2 concentrations from 1-100 nmol/l with the overexpression of MDM2 protein while non-stimulated cells were negative. Stimulation with 1 nmol/l E2 and 10-100 nmol/l EE2 induced MDM2 splicing variants. Hepatocytes treated with 100 nmol/l E2 contained full-length MDM2 mRNA carrying a new type of MDM2 gene mutation. Unstimulated hepatocytes revealed neither mRNA splicing nor alteration of the MDM2 genes. CONCLUSION: The data show that steroid hormones are involved in the induction of MDM2 alterations in benign human hepatocytes. We speculate that some of the alterations may influence MDM2 function, thus possibly favouring genesis of liver changes.

In vitro generation of cytolytic T cells against human melanoma cells overexpressing HDM2.

BACKGROUND: Previous experiments have shown that tumour-associated antigens can be exploited for a successful anti-tumour immunisation. Previous reports demonstrated that oncoprotein MDM2 (HDM2) contains two highly conserved MHC class I binding motifs, MDM2100 and MDM2441, and that dendritic cells (DC) presenting MDM2100 stimulate an effective CTL reaction against melanoma cells. MATERIALS AND METHODS: In this study, we investigated the CTL-inducing capacity of autologous human dendritic cells pulsed with fragment HDM2441. RESULTS: In vitro HDM2441-primed T lymphocytes revealed a strong proliferation activity, released Th-1-associated cytokines, and possessed an effective anti-tumour activity causing apoptosis in HDM2441-overexpressing melanoma cells. Cytotoxic assay demonstrated that in parallel to melanoma cells, up to 65% of primed Tcells also underwent apoptosis. CONCLUSION: These data suggest that HDM2441 may be exploited for broad-spectrum DC-based trials against metastatic melanomas overexpressing HDM2, and point out that the efficacy of such immunotherapeutical approaches may be limited via T cell apoptosis.

Analysis of central regulatory pathways in p53-deficient primary cultures of malignant fibrous histiocytoma exposed to ifosfamide.

Soft tissue sarcomas frequently carry p53 mutations reducing chemotherapeutical response. Especially malignant fibrous histiocytoma (MFH) reveals a reduced ifosfamide (IF) chemosensitivity when compared to other sarcoma entities. This is the first study to analyze MFH cells for the effects of IF on the expression of the pathways P16-CDK4-Rb and P14ARF-MDM2-P73 regulating cell cycle. The aim was to identify candidate genes possibly involved in the anti-apoptotic response of p53-deficient MFH cells during chemotherapy. PCR, real-time RT-PCR and confocal laser scanning microscopy were applied on primary cultures of MFH cells containing defective p53 genes. The cultures were treated with different concentrations of IF. A non-treated MFH culture served as negative control. A threshold concentration of IF (100 microM) was determined sparing the majority of the cells (99%), whereas higher IF quantities caused complete apoptosis. Data collected over a period of 48 h showed that the MFH cells surviving 100 microM IF overexpressed the kinase gene CDK4 and oncogene MDM2 by a factor of 63. A similar strong increase was observed at the protein level for both proteins. In contrast, the other proteins analyzed were not detectable. Additionally, the MFH cells induced complex patterns of MDM2 mRNA splicing and an abnormal mRNA transcript carrying a novel MDM2 missense mutation. These effects were neither observed in the non-treated culture nor in cultures completely inducing spontaneous apoptosis. Therefore, we speculate that the induction of the gene CDK4, and especially of MDM2, is involved in anti-apoptotic mechanisms of p53-negative MFH cells tolerating IF in vitro. Further experiments are necessary to test whether the novel candidate genes favor development of chemoresistance and whether MDM2 mRNA splicing variants contribute to this process in vivo.

Chlamydia trachomatis modulates expression of tumor suppressor gene caveolin-1 and oncogene C-myc in the transformation zone of non-neoplastic cervical tissue.

OBJECTIVES: The obligate intracellular bacterium Chlamydia trachomatis is frequently found in association with benign proliferative, pre-neoplastic and malignant changes in cervical epithelium. The present study addresses the possible role of C. trachomatis infection of the uterine cervix in modulating human cancer gene expression. METHODS: RNA was extracted from both C. trachomatis infected and non-infected human fibroblast cultures treated with ITFgamma. The extracted RNA was used for cDNA microarrays carrying 33,000 human genes to detect abnormal gene expression induced by Chlamydia. Forty specimens of cervix dissected from the transformation zone had previously tested negative for HPV and positive for C. trachomatis by standard DNA PCR (20). These samples were subjected to RT-PCR to detect the expression of the abnormal genes induced by Chlamydia infection. RESULTS: The ITFgamma-induced, non-replicative Chlamydia-infected fibroblast cultures showed significant modulation of gene expression. The cultures showed a 2-fold decrease in the expression of the gene coding for the tumor suppressor caveolin-1, and increased expression of the oncogene C-myc, a promoter of cervical carcinogenesis. In tissues from the Chlamydia-infected cervical transformation zone, real-time RT-PCR demonstrated a highly significant average 4.7-fold reduction of caveolin-1 mRNA (P < or = 0.0001) and an average 2.1-fold increase in C-myc (P < 0.05). CONCLUSIONS: Human ITFgamma-treated fibroblasts as well as non-neoplastic cervical tissues responded to C. trachomatis with a strong down-regulation of caveolin-1 mRNA and a light up-regulation of C-myc mRNA. These changes were independent of the HPV high-risk types. This study reveals possible mechanisms by which C. trachomatis infection may contribute to neoplastic changes in the transformation of uterine cervix. These possible mechanisms require further evaluation.

Anaphylatoxin C5a induces monocyte recruitment and differentiation into dendritic cells by TNF-alpha and prostaglandin E2-dependent mechanisms.

Although monocytes can be directed to develop into dendritic cells (DC) in vitro, the molecular mechanisms that induce their transformation in vivo are largely unknown. In the present study we employed an in vivo SCID mouse model to investigate the impact of two proinflammatory chemotaxins, the anaphylatoxin C5a and the chemokine macrophage inflammatory protein-1alpha (CCL3), on the differentiation of human monocytes and immature DC generated from monocytes in the presence of GM-CSF and IL-4. Both C5a and macrophage inflammatory protein-1alpha recruited human monocytes and immature DC into the peritoneal cavity of SCID mice, but only C5a induced their differentiation into phenotypically mature DC by 48 h after injection. Macrophages derived from monocytes by in vitro culture were resistant to C5a-mediated transformation in vivo. The effect of C5a was indirect, since C5a-stimulated TNF-alpha and PGE(2) were found to be obligatory as well as sufficient to induce differentiation of monocytes. In contrast to monocytes, in vitro generated immature DC required TNF-alpha, but not PGE(2), for their C5a-mediated maturation in vivo. C5a-transformed monocytes represented an inflammatory type of DC, as they constitutively secreted high amounts of TNF-alpha, but also retained the capacity to release the Th1 cytokine IL-12 p70 upon stimulation with CD40 ligand. In summary, we identified for the first time a cascade of inflammatory signals that can induce the transformation of monocytes into DC in vivo. This novel function emphasizes the important immunoregulatory role of C5a at the interface of innate and adaptive immunity.

Down-regulation of Ku 70 and Ku 80 mRNA expression in transitional cell carcinomas of the urinary bladder related to tumor progression.

DNA-dependent protein kinase (DNA-PK) containing the regulatory subunits Ku 70 and Ku 80 plays a prominent role in the repair of double-stranded DNA breaks by a nonhomologous end-joining pathway maintaining genomic stability. In an attempt to elucidate the significance of the DNA-PK complex for human urothelial carcinogenesis, the expression of Ku 70 and Ku 80 was studied in 71 transitional cell carcinomas (TCC) of the urinary bladder of various grades and stages, and in relation to lifestyle and occupational bladder cancer risk factors. To analyse the mRNA expression of Ku 70 and Ku 80, real-time quantitative reverse transcription-polymerase chain reaction was used and the protein expression assessed by immunohistochemistry. Advanced high-grade, high-stage TCC expressed the mRNA of Ku 70 and Ku 80 at a lower level than superficial low-grade, low-stage carcinomas, suggesting down-regulation of the Ku system to be associated with progression of bladder cancer from a low to a high malignant potential. The protein expression of Ku 70 and Ku 80 was closely related and decreased consistently with increasing grades and stages, paralleling the expression of the mRNA. Among hazardous environmental bladder cancer risk factors, heavy consumption of coffee was associated with a twofold decreased Ku 70 and Ku 80 mRNA expression, whereas tobacco smoke did not substantially affect the activity of the Ku system, except for a trend towards a dose-response relationship in the expression of Ku 70 mRNA. There is some evidence that exposure to polycyclic hydrocarbons, paints and lacquer, and stone dust may modify the expression of Ku 70 mRNA. Although the underlying molecular genetic pathways are not yet clearly understood, our data indicate that down-regulation of the Ku system promotes progression of urothelial carcinogenesis to a more malignant and aggressive clinical behavior, presumably as a result of an impaired capacity for DNA repair.

Internalization of cytotoxic analog AN-152 of luteinizing hormone-releasing hormone induces apoptosis in human endometrial and ovarian cancer cell lines independent of multidrug resistance-1 (MDR-1) system.

OBJECTIVE: Eighty percent of human ovarian and endometrial cancers express receptors for luteinizing hormone-releasing hormone (LHRH-R). These receptors can be used for targeted chemotherapy with agents such as AN-152, in which doxorubicin is linked to analog [D-Lys(6)]-LHRH. Direct receptor-mediated antiproliferative effects of AN-152 have been shown in vitro and in vivo. In LHRH-R positive cell lines, AN-152 was more effective than doxorubicin at equimolar concentrations. This study was designed to investigate the mechanism of action of AN-512 in ovarian and endometrial cancer cells in vitro. Study design Three ovarian (SKOV-3, NIH:OVCAR-3, EFO-21) and 2 endometrial carcinoma cell lines (Ishikawa, HEC-1A) were evaluated for doxorubicin- or AN-152-induced apoptosis. Internalization and cytoplasmic release of AN-152 was monitored by confocal laser scanning microscopy and inhibited by chloroquine. Cleavage of doxorubicin from AN-152 was inhibited by carboxylesterase inhibitor, diisopropyl fluorophosphate (DFP). The surface expression of multidrug resistance-1 (MDR-1) gene product P-glycoprotein (Pgp) was measured by flow cytometry. RESULTS: Induction of apoptosis by AN-152 in LHRH-R positive Ishikawa, HEC-1A, EFO-21, and NIH:OVCAR-3 cells was significantly higher than that induced by doxorubicin, whereas the percentage of apoptotic cells in LHRH-R negative SKOV-3 was higher after treatment with doxorubicin. In EFO-21 cells, apoptosis induced by AN-152 was inhibited by pretreatment with chloroquine. Pretreatment with DFP increased AN-152-induced apoptosis in LHRH-R positive cells and reduced apoptosis in LHRH-R negative SKOV-3. Both AN-152 and doxorubicin induced surface expression of MDR-1 gene product Pgp, but the effect of AN-152 was smaller than that of doxorubicin. Pgp surface expression induced by AN-152 was inhibited by pretreatment with DFP. CONCLUSION: AN-152 is internalized through the LHRH-R and induces apoptosis in LHRH-R-positive human ovarian and endometrial cancer cell lines without activating the MDR-1 efflux pump system. The efficacy and specificity of AN-152 is inversely correlated with carboxylesterase activity.