Cre recombinase-mediated inactivation of H-2Dd transgene expression: evidence for partial missing self-recognition by Ly49A NK cells.

We have established H-2D(d)-transgenic (Tg) mice, in which H-2D(d) expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2D(d) Tg in H-2(b) mice and acquired reactivity to cells lacking H-2D(d), both in vivo and in vitro. H-2D(d)-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2D(d) expression. That abrogated NK cell reactivity to cells lacking D(d). In D(d) single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking D(d), because the inhibitory Ly49A receptor is no longer engaged by its D(d) ligand. In contrast, Ly49A+ NK cells from D(d) x MxCre double Tg mice were unable to react to D(d)-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated D(d) expression thus impacts specifically missing D(d) but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of D(d) from some host cells results in the acquisition of only partial missing self-reactivity.

Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies.

Cancer cell lines are widely used in many types of cancer research, including studies aimed at understanding DNA hypermethylation of gene promoters in cancer. Hypermethylation of promoters is capable of repressing the expression of tumor suppressor genes and may play a role in the development and/or progression of cancer. Although both primary malignancies and cancer cell lines exhibit this epigenetic phenomenon, there has been no direct comparison between them. In order to address this question, we have utilized restriction landmark genomic scanning to measure the hypermethylation phenotypes of cancer cell lines and compared these data with the same analysis performed on primary malignancies. In all cases, cancer cell lines exhibit significantly higher levels of CpG island hypermethylation than the primary malignancies they represent. Colon cancer cell lines are most similar to their respective tumors, with only a 5-fold increase in hypermethylation, while head and neck squamous cell carcinoma cell lines show a 93-fold increase in hypermethylation. Furthermore, >57% of the loci methylated in cell lines are never methylated in 114 primary malignancies studied. Seventy percent of loci hypermethylated in cell lines are hypermethylated in lines from more than one type of cancer. These data indicate that most CpG island hypermethylation observed in cancer cell lines is due to an intrinsic property of cell lines as opposed to the malignant tissue from which they originated.

Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci.

Aberrant DNA methylation is believed to be important in tumorigenesis by causing either transcriptional inactivation of genes or chromosomal instability. Several laboratories have identified promoter hypermethylation of tumor suppressor genes in acute myeloid leukemia (AML). However, these studies do not provide a global assessment of overall methylation changes and do not allow the identification of novel methylated sequences. Previously, nonrandom CpG island methylation was reported in 17 adult de novo AML diagnostic samples when compared with the corresponding remission samples by means of restriction landmark genomic scanning (RLGS). That study has been expanded on by an analysis of a larger set of CpG islands (1740 vs 1184), which now provides details of 33 cloned methylated loci, including 21 known genes or expressed sequence tags. Five of these cloned loci appear to be methylated only in AML and not in the 6 solid tumors studied in this study (more than 98 samples analyzed). Chromosomal location was available for 30 of the 33 loci, and 5 of these 30 (17%) are localized to chromosome 11, suggesting a trend toward overrepresentation of methylation events on this chromosome. These results provide evidence for widespread aberrant methylation in AML, with identification of novel methylation targets, epigenetic changes that appear unique to AML, and apparent preferential methylation on chromosome 11.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Identification of a novel member of the snail/Gfi-1 repressor family, mlt 1, which is methylated and silenced in liver tumors of SV40 T antigen transgenic mice.

DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.

Transgenic expression of Ly49A on T cells impairs a specific antitumor response.

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.

Restriction landmark genomic scanning of mouse liver tumors for gene amplification: overexpression of cyclin A2.

SV40 T/t antigen-induced liver tumors from transgenic mice were analyzed by Restriction Landmark Genomic Scanning (RLGS). Using NotI as the restriction landmark, RLGS targets CpG islands found in gene-rich regions of the genome. Since many RLGS landmarks are mapped, the candidate gene approach can be used to help determine which genes are altered in tumors. RLGS analysis revealed one tumor-specific amplification mapping close to CcnA2 (cyclin A2) and Fgf2 (fibroblast growth factor 2). Southern analysis confirmed that both oncogenes are amplified in this tumor and in a second, independent liver tumor. Whereas Fgf2 RNA is undetectable in tumors, CcnA2 RNA and cyclin A2 protein was overexpressed in 25 and 50% of tumors, respectively. Combining RLGS with the candidate gene approach indicates that cyclin A2 amplification and overexpression is a likely selected event in transgenic mouse liver tumors. Our results also indicate that our mouse model for liver tumorigenesis in mice accurately recapitulates events observed in human hepatocellular carcinoma.

Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1.

NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development.

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.

[Basic tenets for quality circles in North Rhine Medical Service of Public Health Insurance]. / Grundsätze für Qualitätszirkel beim MDK Nordrhein.

The Medical Advisory Service of the Health Insurance in the area of the Northern Rhine (MDK Nordrhein) has set up an internal concept for quality management since 1998. This concept includes the installation and performance of quality circles. Staff members were internally qualified as "presenters". They worked out principles for quality circles of the MDK North Rhine which were implemented as binding basic rules by the managing conference of the MDK. The principles will be presented in detail.

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Methylation and downregulated expression of mac25/insulin-like growth factor binding protein-7 is associated with liver tumorigenesis in SV40T/t antigen transgenic mice, screened by restriction landmark genomic scanning for methylation (RLGS-M).

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis. An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5' flanking region and 5' portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5' to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene.

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.

Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells.

The identity of cells that mediate positive selection of CD8(+) T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated beta(2)-microglobulin mutant mice or mice with mutations in both the K(b) and D(b) genes were repopulated with fetal liver cells from class I(+) TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8(+) T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8(+) T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8(+) T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)-TCR(+) CD8(+) T cells was observed in class I(+) into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed.

[ENT expert assessment for federal health insurance in North Rhine. A contribution to the internal discussion of quantitative and qualitative optimization of the expert witness process]. / HNO-Begutachtung im MDK Nordrhein. Ein Beitrag zur internen Diskussion über die quantitative und qualitative Optimierung eines Begutachtungsprozesses.

Contribution to an Internal Discussion on Quantitative and Qualitative Optimisation of Expertising Procedures: The medical advisory and expertising Services of the German statutory sickness insurance are according to German social legislation authorised to request supporting documents direct from the treating physicians or hospitals. The consultant medical expertiser for ENT diseases at Cologne worked out the percentage of requests that resulted in "further investigations" and the amount of work involved. A document form was used to record all such requests for a given period. 154 expertising procedures concerning ENT diseases were counted within a period of 5 weeks. 47% of the patients concerned pertained to workers' sickness insurance funds and 29% to guilds sickness insurance funds. Two thirds of all requests were channelled through the statutory sociomedical preconsultant service and one-third were sent by post. 55% of all the requested documents were considered to be "complete" and 45% "incomplete". N = 100 requests were initial and N = 54 were follow-up requests. The answers received were considered satisfactory and final in 62% of the requests, whereas "further inquiries" were considered necessary in n = 58 (38%) of the cases. 80% of the cases in which the requests resulted in satisfactory information, were approved for sickness insurance payment and approximately 20% were rejected. The average period of time that elapsed between a request for more information and the reply and the final expertising given to and passed on to the patient by the statutory insurance body was 2 weeks. In cases where "further inquiries" are considered necessary, three additional working days are usually required in the Cologne ENT diseases area. If the statutory sociomedical preconsultant service procedure could be intensified, this would result in better channelling of requests by the ENT medical and advisory service of the stautory sickness insurance bodies. A more pronounced examination by the statutory sociomedical preconsultant service could minimise the need for additional requests, follow-ups and "further inquiries" and could increase the speed of completion of approved or rejected insurance cases from 14 days to at the most one day or even 0 days. This pilot study was conducted to encourage the local bodies to optimise their work. In the long run good quality of work, patient satifaction and patients' job security will result and will be the corner-stones of modern quality assurance and quality management in North-Rhine Westphalian medical expertising and consultant services in association with the relevant statutory sickness insurance bodies.

Restriction landmark genome scanning for aberrant methylation in primary refractory and relapsed acute myeloid leukemia; involvement of the WIT-1 gene.

There is substantial evidence to suggest that aberrant DNA methylation in the regulatory regions of expressed genes may play a role in hematologic malignancy. In the current report, the Restriction Landmark Genomic Scanning (RLGS) method was used to detect aberrant DNA methylation (M) in acute myeloid leukemia (AML). RLGS-M profiles were initially performed using DNA from diagnostic, remission, and relapse samples from a patient with AML. Rp18, one of the eight spots found that was absent in the relapse sample, was cloned. Sequence analysis showed that the spot represented a portion of the WIT-1 gene on human chromosome 11p13. Rp18 was missing in the relapse sample due to a distinct DNA methylation pattern of the WIT-1 gene. Twenty-seven AML patients that entered CR after therapy (i.e., chemosensitive) were studied and only 10 (37%) of the diagnostic bone marrow (BM) samples showed methylation of WIT-1. However, seven of eight (87.5%) diagnostic BM samples from primary refractory AML (chemosensitive) showed methylation of WIT-1. The incidence of WIT-1 methylation in primary refractory AML was significantly higher than that noted in chemosensitive AML (P=0.018). Together, these results indicate that RLGS-M can be used to find novel epigenetic alterations in human cancer that are undetectable by standard methods. In addition, these results underline the potential importance of WIT-1 methylation in chemoresistant AML.

[Quality assurance and teamwork in social medicine expert assessment exemplified by the expert assessment aid "corrective breast surgery" of the North Rhine Health Insurance Medical Service]. / Qualitätssicherung und Mitarbeiterpartizipation in der Sozialmedizinischen Begutachtung am Beispiel der Begutachtungshilfe "korrektive Mamma-Chirurgie" des MDK Nordrhein.

To standardise sociomedical expertising procedures in surgery of the breast a project team of medical advisors of the Medical Advisory and Expertising Service North Rhine worked out a manual. A form was created for sociomedical pre-advice occasions. Detail parameters for examination (time needed and advisor selection) were fixed in consent. Checklists were worked out, which may be admitted to quality assurance procedures. The manual "Medical opinion for surgery of the breast" has been implemented and is applied in the Medical Advisory and Expertising Service North Rhine since January 1999. In the sense of teamwork participation those who were involved as team-workers became participants in decision-making. The manual "Medical opinion for surgery of the breast" may be seen as an example of quality assurance and participation in socio-medical opinion procedures. The influence of this manual on the formal quality of medical opinion will be checked.

Genomic imprinting and Igf2 influence liver tumorigenesis and loss of heterozygosity in SV40 T antigen transgenic mice.

Maternal-specific loss of heterozygosity (LOH) and allelic imbalances [i.e., partial LOH (pLOH)] observed in SV40 T/t antigen-induced liver tumors suggests that an imprinted gene on chromosome 7 is involved in liver tumorigenesis. Maternal-specific LOH/pLOH may reflect the loss of a maternally expressed tumor suppressor gene or the acquisition of paternally active alleles of a growth promoter. In addition, two oppositely imprinted genes on distal chromosome 7, Igf2 and H19, are re-expressed in most liver tumors from an SV40 T/t antigen transgenic line (M11T-G). Igf2 is a paternally expressed growth promoter, and H19 is a maternally expressed gene that can suppress growth in some tumor cell lines. We studied the role of Igf2 during liver tumorigenesis by creating Igf2 (+/-) M11T-G mice. These mice are essentially null for Igf2 expression because imprinting normally precludes maternal Igf2 expression. M11T-G, Igf2 (+/-) males exhibit a 15-fold reduction in the frequency of large tumors. Igf2 (+/-) tumors do not express maternal Igf2, indicating rigid imprinting control in the liver. LOH/pLOH analysis was performed on the tumors and indicates that acquisition of paternally active Igf2 alleles is a major selective event for M11T-G liver tumorigenesis. This also implies the existence of an imprinted, maternally expressed tumor suppressor gene on chromosome 7 that is unlikely to be H19.

Restriction landmark genomic scanning (RLGS-M)-based genome-wide scanning of mouse liver tumors for alterations in DNA methylation status.

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect, and subsequently clone, genomic regions with alterations in DNA methylation associated with tumorigenesis. Use of a methylation-sensitive enzyme for the landmark cleavage allows analysis of changes in methylation patterns. In this study, we used RLGS-M to analyze SV40 T antigen-induced mouse liver tumors derived from interspecific F1 hybrids between Mus spretus (S) and C57BL/6 (B6). Because 575 S- and B6-specific RLGS loci/spots have been mapped, tumor-related alterations in the RLGS profile could be immediately localized to specific chromosomal regions. We previously found that the loss of contiguous loci/spots could be attributed primarily to DNA loss, whereas loss of solitary loci/spots could be attributed primarily to DNA methylation. In this study, we examined 30 mouse liver tumor samples for loss of the 507 mapped loci/spots. Fourteen solitary loci/spots found to be absent or reduced in more than 75% of tumor samples were cloned and subjected to DNA sequence analyses. Two loci were identified as alpha4 integrin and p16/CDKN2, genes reported to be involved in tumorigenesis. Thus, RLGS-M can detect alterations in the methylation status of known tumor suppressor genes and provide a method for detecting and subsequently cloning novel genomic regions that undergo alterations in methylation during tumorigenesis.