Identification of the pathogenic pathways in osteoarthritic hip cartilage: commonality and discord between hip and knee OA.

OBJECTIVE: To define for the first time the transcriptomes of normal and end-stage osteoarthritis (OA) hip cartilage. MATERIALS AND METHODS: RNA was isolated from cartilage within 2h of joint replacement surgery. Gene expression was analyzed using Agilent GeneSpring GX 11 following hybridization to Illumina Human HT-12 V3 microarrays. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to validate the expression of six genes identified by microarray as differentially expressed. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) were used to investigate enriched functions or canonical pathways amongst differentially expressed genes respectively. RESULTS: In total we identified 998 differentially expressed genes (fold change ≥ ±1.5, P-value ≤ 0.01) between neck of femur fracture (NOF) (n = 10) and OA hip (n = 9) patient cartilage. These differentially expressed genes were enriched within 71 canonical pathways. A comparison between a comparable knee dataset(20) only identified 229 genes similarly differentially expressed although remarkably 34 canonical pathways overlapped between experiments. CONCLUSIONS: This study is the first to report a comprehensive gene expression analysis of human hip OA cartilage compared to control (NOF) cartilage at the whole-genome level. Our differential gene expression dataset shows excellent correlation with similar defined studies using comparable tissue but reveals discord between hip and knee OA at the individual gene status but with commonality with regards the molecular pathways involved.

Pattern recognition receptor expression is not impaired in patients with chronic mucocutanous candidiasis with or without autoimmune polyendocrinopathy candidiasis ectodermal dystrophy.

Patients with chronic mucocutaneous candidiasis (CMC) have an unknown primary immune defect and are unable to clear infections with the yeast Candida. CMC includes patients with AIRE gene mutations who have autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and patients without known mutations. CMC patients have dysregulated cytokine production, suggesting that defective expression of pattern recognition receptors (PRRs) may underlie disease pathogenesis. In 29 patients with CMC (13 with APECED) and controls, we assessed dendritic cell (DC) subsets and monocyte Toll-like receptor (TLR) expression in blood. We generated and stimulated monocyte-derived (mo)DCs with Candida albicans, TLR-2/6 ligand and lipopolysaccharide and assessed PRR mRNA expression by polymerase chain reaction [TLR-1-10, Dectin-1 and -2, spleen tyrosine kinase (Syk) and caspase recruitment domain (CARD) 9] in immature and mature moDCs. We demonstrate for the first time that CMC patients, with or without APECED, have normal blood levels of plasmocytoid and myeloid DCs and monocyte TLR-2/TLR-6 expression. We showed that in immature moDCs, expression levels of all PRRs involved in anti-Candida responses (TLR-1, -2, -4, -6, Dectin-1, Syk, CARD9) were comparable to controls, implying that defects in PRR expression are not responsible for the increased susceptibility to Candida infections seen in CMC patients. However, as opposed to healthy controls, both groups of CMC patients failed to down-regulate PRR mRNA expression in response to Candida, consistent with defective DC maturation, as we reported recently. Thus, impaired DC maturation and consequent altered regulation of PRR signalling pathways rather than defects in PRR expression may be responsible for inadequate Candida handling in CMC patients.

Expression and chromosomal localization of the cytochrome P1-450 gene in human mitogen-stimulated lymphocytes.

Genetic differences in aryl hydrocarbon hydroxylase (AHH) (flavoprotein-linked monoxygenase EC 1.14.14.1) activity in cultured lymphocytes have been linked with individual risk for certain environmentally caused cancers. Cytochrome P1-450 is the form of cytochrome P-450 most closely associated with AHH activity. In this study the chromosomal localization and the expression of human cytochrome P1-450 gene were determined in phytohemagglutinin-stimulated lymphocytes. In situ hybridization analysis provides assignment of the structural gene for human cytochrome P1-450 to chromosome 15q22-q24. Treatment of lymphocytes with benzanthracene increased the amount of mRNA hybridized to the cloned cytochrome P1-450 gene. The level of cytochrome P1-450 mRNA in these lymphocytes correlates well with the induced AHH activity indicating that non-cytochrome P1-450 enzymes contribute little to the individual differences in the level of AHH activity in the lymphocytes. Southern analyses of genomic DNA from individuals with high and low induced AHH activity demonstrated no detectable differences in the pattern or intensity of restriction fragments after treatment with benzanthracene from either individual. This finding together with the excellent correlation between the induced cytochrome P1-450 and AHH activity, suggests that transcriptional control rather than gene amplification or gross form of gene rearrangement accounts for cytochrome P1-450 induction in man. Measurements of cytochrome P1-450 mRNA content in cultured lymphocytes provide an alternative approach to the assay of AHH activity in assessing AHH phenotype and predicting different susceptibilities to deleterious environmental agents.

Studies of myeloperoxidase gene expression at the cellular level by in situ hybridization.

Recent studies have demonstrated myeloperoxidase (MPO) gene expression during granulocytic differentiation. Since these studies have been done exclusively by Northern and dot blot analysis and frequently with mixed populations of cells, quantitative changes in gene expression for particular populations of cells are difficult to assess. We therefore examined MPO expression at the cellular level in various normal and malignant hematopoietic cells by the in situ hybridization (ISH) technique. Using this approach, we demonstrated that inducing the promyelocytic HL-60 cell line to differentiate along either monocytic or granulocytic pathways decreases MPO mRNA expression. Similarly, when ISH was performed on normal bone marrow, relatively high levels of MPO mRNA were detected in myeloblasts, promyelocytes, and early eosinophilic precursors, whereas the expression was markedly decreased in more advanced stages of myeloid differentiation. These findings agree with the known decrease in MPO protein synthesis observed during granulocytic differentiation and suggest that regulation of MPO protein synthesis occurs at the level of MPO mRNA expression. We conclude by showing that ISH can detect MPO mRNA in myeloblasts of patients with acute leukemia and can be a potentially useful technique in the study of myeloid differentiation in acute leukemias.

Association of human papillomavirus with penile carcinoma: a study using polymerase chain reaction and in situ hybridization.

Although carcinoma of the uterine cervix has been shown to be strongly associated with human papillomavirus (HPV) infection, a sexually transmitted disease, similar detailed data are lacking in penile carcinoma. To determine the association of HPV with penile carcinoma, we examined 30 specimens of penile carcinoma from 23 patients by the highly sensitive polymerase chain reaction (PCR) and in situ hybridization assays. We also examined nonneoplastic penile foreskins from 20 adults using the polymerase chain reaction assay. Human papillomavirus type 16 genome was found in 15 patients (65%), HPV type 30 was found in three (13%), and HPV type 6/11 was found in two (9%). These HPV types were not detected in any of the nonneoplastic foreskins. As in cervical carcinoma, HPV, particularly type 16, is strongly associated with penile carcinoma and may play an etiologic role in the development of this neoplasm.

p53 mutations in human bladder cancer.

Mutations in the tumor suppressor gene p53 play an important role in carcinogenesis and tumor progression. To assess the status of p53 from genomic DNA from bladder cancer samples a two stage polymerase chain reaction was employed. The technique provided material for subsequent detection of mutations by Single Strand Conformation Polymorphism (SSCP) analysis followed by DNA sequence analysis. SSCP analysis of exons 5 to 9 of p53 was performed using fragments from PCR end-labeled with 32P followed by autoradiography using an electrophoresis system with temperature control. This SSCP method improved resolution of mutations in exons 5, 7, and 8 and the sharpness of bands in exons 6 and 9. Bands with altered migration patterns were excised from the dried SSCP gels, reamplified by PCR, and sequenced. Mutations in conserved exons 5, 6, 7, 8, and 9 of the p53 gene were analyzed from bladder tumor biopsies. Our results are consistent with the literature in that mutations in p53 are predominantly found in high grade bladder cancer (Odds Ratio = 4.05, Fisher Exact P = 0.104); however, the results were not statistically significant due to small numbers. Eight of 35 (23%) tumor samples examined showed mutations in p53 (including two double mutations). Six of 13 (46%) grade III and IV tumors had p53 mutations vs. 2 of 17 (12%) grade I and II tumors. Normal individuals carried no p53 mutations. We found no correlation between pack years of smoking and mutation in p53. The spectrum of mutations confirmed a high proportion of G:C C:G transversions as well as the occurrence of double mutations.

Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18.

BACKGROUND: Quantitation of human papillomavirus (HPV) DNA in clinical samples may yield important clinical information. METHODS AND RESULTS: We developed a 5' exonuclease fluorescent probe assay for HPV quantitation that uses real-time PCR. The assay was optimized for HPV types 6 (HPV-6), -11, -16, and -18. A multiplex format was developed to quantify a cellular target of known iteration simultaneously with HPV quantitation, which controls for the amount of input DNA. Dilution series of target and heterologous templates were used to verify the assay. The assay was successfully used on fresh and PreservCyt-fixed cell lines, as well as cervical samples. The linear range of the assay is from 10 to 10 million copies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures. CONCLUSION: The method is accurate over a large copy number range, reproducible, type specific, normalized for input DNA quantity, and applicable to PreservCyt-fixed material.

A case-control study of human papillomavirus and cervical squamous intraepithelial lesions (SIL) in Harris County, Texas: differences among racial/ethnic groups.

We conducted a case-control study of the association between SIL and HPV among whites (W), African Americans (AA), and Hispanics (H) in Harris County, Texas. Cases were identified at M.D. Anderson Cancer Center Colposcopy Clinic. Controls were identified among women obtaining routine Pap screening at two Harris County Health Department Clinics. HPV was detected by a PCR-based fluorescent assay. Dichotomous and polytomous logistic regression models were used to estimate adjusted odd ratios (AOR) and 95% confidence intervals (CI) for SIL among racial/ethnic groups and grade of disease. Prevalence of HPV infection was 64% in low grade SIL (LSIL), 84% in high grade SIL (HSIL), and 19% in controls. Risk of SIL was higher in H than in W and AA, AOR 29.5 (12.4-70.5), 15.3 (6.0-33.8), and 5.8 (2.6-12.6), respectively. Similarly, racial/ethnic differences were observed for both LSIL (AOR = 16.6, 7.7, and 4.3, respectively) and HSIL (AOR = 78.6, 34.6, and 14.2, respectively). Findings support the association between SIL and HPV and differences in the strength of the association with LSILs and HSILs. Data also suggest a higher risk for H and a lower risk for AA.

Comparative genome hybridization of Streptococcus mutans strains.

The basis for genotypic and phenotypic variation within Streptococcus mutans is poorly understood but the availability of the genome sequence of strain UA159 provides an opportunity for comparative studies. Genomic DNA prepared from nine strains of S. mutans was used to probe a microarray consisting of oligonucleotides representing 1948 open reading frames of S. mutans UA159. A total of 385 (20%) of the UA159 open reading frames were found to be absent from one or more of the test strains. Absent open reading frames frequently occurred in blocks of adjacent open reading frames and represented regions previously experimentally detected by polymerase chain reaction, predicted genomic islands and insertion sequence elements as well as novel open reading frames. Approximately half appear to involve foreign DNA acquired by horizontal transmission. The results indicate the existence of distinct core and dispensable genomes and may help explain the phenotypic and genotypic variation within S. mutans.

Cellular proteins involved in papillomavirus-induced transformation.

Human papillomaviruses (HPVs) are associated with at least 80% of cervical carcinomas and are classified as high-risk or low-risk based on whether or not they are commonly found in cervical cancers. The high-risk HPVs have early gene products (E6 and E7) that immortalize human keratinocytes and are at least partially responsible for causing cervical carcinoma. E6 and E7 from the high-risk viruses interact strongly with the tumor suppressors p53 and Rb; those from the low-risk HPVs do not. Transformation involves a multi-step process and requires additional factors besides high-risk HPV infection. High-risk HPVs are capable of immortalizing primary human keratinocytes in tissue culture, but such cells become transformed only after certain chromosomal changes take place, possibly having to do with oncogene activation. The DNA of high-risk HPVs is frequently (if not always) integrated into the genome of cancer cells; it is normally episomal in premalignant lesions. Integration disrupts the E2 and E5 genes and viral gene regulation. Cells containing integrated viral DNA show excessively high levels of E6 and E7. While there is some conflicting evidence, it appears that the p53 and Rb tumor-suppressor genes are more frequently mutated in HPV-negative tumors than they are in HPV-positive tumors, suggesting that for tumor formation to proceed the p53 and Rb proteins must be inactivated either by interaction with the viral proteins or by mutation. The presence of an activated oncogene in a cell lacking functional p53 or Rb may then be sufficient to cause tumor progression.

Isolation and characterization of a cloned DNA sequence associated with the murine Ah locus and a 3-methylcholanthrene-induced form of cytochrome P-450.

Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P(1)-450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of Escherichia coli DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by Pst I cleavage and homopolymeric "tailing" and cloned in E. coli LE392. Clone 46 hybridized with [(32)P]cDNA made from 23S mRNA from "Ah-responsive" C57BL/6N mice but did not hybridize with similarly prepared [(32)P]cDNA from "Ah-nonresponsive" DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [(32)P]cDNA probes; these two clones were therefore used as "positive" and "negative" control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti-P(1)-450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of Pst I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal Pst I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of Pst I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two (Pst I and Xba I) had sites within the clone 46 DNA insert. After hybridization of clone 46 (32)P-labeled nick-translated DNA to EcoRI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine Ah locus, presumably the structural gene encoding 3-methylcholanthrene-induced P(1)-450.

A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA.

A simple method for the detection of a number of human papillomavirus (HPV) genotypes associated with cervical cancer has been developed. The assay exploits the 5'-->3' exonucleolytic activity of Taq DNA polymerase to increase the signal from fluorescent dyes by releasing them from genotype-specific probes during PCR. The probes are oligonucleotides with a 5' reporter dye (6-carboxyfluorescein), a quencher dye (6-carboxy-tetramethyl-rhodamine), and a phosphate-blocked 3' end. In the intact probe, the proximity of the reporter and the quencher results in suppression of reporter fluorescence by Förster-type energy transfer (V. T. Förster. Ann. Phys. 2:55-75, 1948). If the probe is bound downstream of either primer during PCR, the 5'-->3' exonucleolytic activity of Taq polymerase degrades it, allowing the reporter to diffuse away from the quencher, which results in an increase in reporter fluorescence. The increased fluorescence is directly related to the amount of target DNA and can be detected with an automated fluorometer. Probes for the L1 region of the cervical-cancer-associated HPV types 16, 18, 31, 33, and 35 were synthesized and the assays were optimized. The most sensitive assay can detect as few as two copies of HPV DNA in human cervical specimens.

Induction of chromosome banding by trypsin/EDTA for gene mapping by in situ hybridization.

We describe an easy and reproducible procedure that utilizes trypsin/EDTA for the induction of chromosome banding in conjunction with in situ hybridization. The high quality banding resolution required for grain localization is obtained on both elongated and contracted chromosomes derived from synchronized or nonsynchronized human lymphocytes or fibroblasts. This procedure can also be useful for gene localization on chromosomes from cancer cells.

Immunoglobulin synthesis and gene rearrangements in lymphoid cells transformed by replication-competent Rauscher murine leukemia virus: transformation of B cells at various stages of differentiation.

Lymphoid cells transformed by Rauscher murine leukemia virus (R-MuLV) belonged to the B cell lineages. One group of cells exhibited Fc receptors but completely lacked immunoglobulin mu heavy and kappa light chains. The majority of the cells resemble pre-B type. They displayed mu chains but kappa chains were completely absent. Very rarely certain cells synthesized both mu and kappa chains. Based on the presence of Fc receptors and IgM synthesis the cells transformed by R-MuLV belonged to three B cell developmental stages. These cells were tested for immunoglobulin gene rearrangements using JH and CK probes. DNA from cell lines without any detectable levels of IgM mu exhibited embryonic as well as rearranged JH genes, whereas cells expressing IgM possess, in addition, productive and non-productive light chain gene rearrangements. The most terminally differentiated cell possesses JH and CK rearrangement associated with the synthesis of mu and kappa chains. Presumably the cells with rearranged JH and CK genes without immunoglobulin synthesis represent a developmental transition. We conclude that cells transformed by R-MuLV belonged to five step-wise compartments of B cell development. Our findings implicate definite sequential events of immunoglobulin gene rearrangement and expression during B cell development.

Localization of human variable and constant region immunoglobulin heavy chain genes on subtelomeric band q32 of chromosome 14.

Analysis of a group of human/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human variable region (VH), junctional (JH), and constant region (C epsilon) heavy chain immunoglobulin genes indicates that all of these IgH genes are localized on the subtelomeric (q32) band of chromosome 14. Somatic cell hybrids were isolated in selective medium after fusing human fibroblasts with hprt- Chinese hamster cells. The human parental cells contained two translocation chromosomes representing a reciprocal translocation between chromosomes X and 14. Only those hybrid cell lines retaining a complete human autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32) retained the human IgH genes. Retention of these genes did not correlate with the presence of the other translocation chromosome, 14/X. These results indicate that all human IgH genes (VH, JH, and CH) map to the same chromosomal band (14q32) which is commonly involved in reciprocal translocations with human chromosome 8 (8q24) in B-cell neoplasms.

Chromosomal localization of three human ras genes by in situ molecular hybridization.

Three human ras family protooncogenes, c-Ki-ras-1, and c-Ki-ras-2, and N-ras, have been mapped to chromosome bands 6p11-12, 12p11.1-12.1, and 1p11-13, respectively by in situ molecular hybridization. Certain human cancers display consistent and specific alterations involving chromosomes 1, 6, and 12. The precise chromosomal localization of ras genes will permit evaluation of the possible effect of these chromosome changes on the structure and activities of ras protooncogenes in human neoplasia.

Chromosomal mapping of the simian sarcoma virus onc gene analogue in human cells.

The primate cell-derived transforming gene (v-sis) of simian sarcoma virus (SSV) is represented as a single copy marker within cellular DNAs of mammalian species including human. The human analogue of v-sis can be distinguished from its rodent counterparts by Southern blotting analysis of EcoRI-restricted DNAs. By testing for the presence of the human v-sis-related fragment, c-sis (human), in somatic cell hybrids possessing varying numbers of human chromosome, as well as in segregants of such hybrids, it was possible to assign c-sis to human chromosome 22.

Molecular cloning and chromosomal mapping of a human locus related to the transforming gene of Moloney murine sarcoma virus.

Human DNA was analyzed for the presence of sequences homologous to the transforming gene (v-mos) of Moloney murine sarcoma virus. A single 2.5-kilobase pair (kbp) EcoRI-generated fragment of human DNA was identified by using cloned v-mos as probe. This DNA was molecularly cloned in a bacteriophage vector. By heteroduplex and restriction enzyme analyses, this human DNA fragment, designated c-mos (human), contained a 0.65-kbp region of continuous homology with v-mos and was present as a single copy in human DNA. By testing for the presence of c-mos (human) in somatic cell hybrids possessing various numbers of human chromosomes, as well as in subclones of such hybrids, it was possible to assign c-mos (human) to human chromosome 8.

Localization of the normal allele of T24 human bladder carcinoma oncogene to chromosome 11.

The development of DNA-mediated gene transfer techniques has made it possible to identify transforming genes present in certain human tumour cells. Such genes have been shown to induce morphological transformation when used to transfect suitable assay cells. Recently a transforming gene has been isolated by molecular cloning techniques from the T24 (ref. 11) and EJ (ref. 12) human bladder carcinoma cell lines. This bladder carcinoma oncogene has been shown to be of human origin, less than six kilobase pairs (kbp) in size, and closely related to the onc genes (v-bas and v-ras) of BALB and Harvey murine sarcoma viruses. These transforming retroviruses arose in nature by transduction of cellular genes from mouse and rat cells, respectively. To understand better the relationship of the T24 oncogene with other human cellular genes, we have determined the chromosomal location of its normal allele within the human genome. We show here that it is carried on chromosome 11 in normal cells.