Molecular analyses of chromosome 12 in chronic lymphocytic leukemia.

Trisomy 12 is the most common chromosomal aberration in chronic B lymphocytic leukemia (B-CLL). In this study we have investigated trisomy 12 and posed two major questions: (a) What is the origin of the third copy of chromosome 12? and (b) What is the proportion of trisomy 12 cells in malignant clones with this aberration? The origin of an extra copy of chromosome 12 in lymphocytes from patients with B-CLL was studied by the use of probes detecting restriction fragment length polymorphisms on this chromosome. In all six patients that were evaluable, the third copy was derived from a simple duplication of one of the original chromosomes. In none of these patients nor in four patients with two copies of chromosome 12 were losses of the homologue observed. When studying metaphase cells from some CLL patients with trisomy 12, a large proportion of the cells are found to have a normal karyotype. In this study the fraction of normal metaphases was not matched by a similar fraction of cells lacking trisomy 12, as judged by scanning densitometry of hybridization bands. Thus, normal metaphases appear to be derived from a small fraction of easily stimulated probably nonmalignant cells and not from a large second population of malignant cells with a normal karyotype.

No evidence of trisomy 12 or t(11;14) by molecular genetic techniques in chronic lymphocytic leukemia cells with a normal karyotype.

Chromosome analysis of B-cell mitogen-activated cells from patients with B-cell chronic lymphocytic leukemia (CLL) show clonal abnormalities in approximately one half of the cases. Among the most frequent aberrations are trisomy 12 and t(11;14). In the other half, no clonal chromosomal abnormalities are found. We wished to determine whether these CLL clones are cytogenetically normal or whether the apparently normal karyotype results from an inability of the malignant cells to enter metaphase in vitro, leaving only the few remaining normal cells to be karyotypically studied. Probes that can detect restriction fragment length polymorphisms (RFLPs) on chromosome 12, which makes it possible to determine the copy number of this chromosome, as well as bcl-1 probes that can detect t(11;14) were used. Of 13 CLL cases with normal karyotypes, none showed evidence for either trisomy 12 or t(11;14). This finding indicates that CLL cases with an apparently normal karyotype do not have two of the most common clonal chromosomal aberrations and may be karyotypically normal. The study also shows that Southern blot analyses, using probes that can detect RFLPs on chromosome 12, can be used for rapid and simple detection of trisomy 12 in CLL patients.

Effects by 8-bromo-cyclicAMP on basal and organic dust-induced release of interleukin-6 and interleukin-8 in A549 human airway epithelial cells.

Inhalation of organic dust from a swine-confinement building leads to an intense inflammatory reaction with an increased number of inflammatory cells and mediators in the upper and lower respiratory tract of previously unexposed subjects. In vitro the dust induces cytokine release from epithelial cells and alveolar macrophages. It is known that intracellular cyclic AMP (cAMP) contributes to the regulation of inflammatory responses. We therefore investigated whether 8-Bromo-cAMP, a cell membrane-permeable cAMP analogue, would influence release of the cytokines interleukin-6 (IL-6) and IL-8 in a human airway epithelial cell line, A549, exposed to a suspension of the organic dust, and to a supernatant prepared by centrifugation (at low g-force) of a suspension of dust. The large particulate matter was thus sedimented, leaving bacteria, whole and cell wall constituents in the supernatant. Cytokine release was measured with enzyme-linked immunosorbent assay (ELISA). The cytokine release induced by a supernatant was 23% (IL-6) and 27% (IL-8) of the release induced by a dust suspension. 8-Bromo-cAMP (1 mM) doubled basal IL-6 release and IL-6 release induced by a dust supernatant (P<0.01), and increased IL-6 release induced by a dust suspension by 19% (P<0.05). 8-Bromo-cAMP did not affect basal IL-8 release, partially inhibited (28%) the release of IL-8 induced by a dust suspension (P<0.01), but increased IL-8 release induced by a dust supernatant by 13% (P<0.05). In summary, expression of the cytokines IL-6 and IL-8 is differentially regulated by 8-Bromo-cAMP, both with regard to basal and dust-induced release. The results indicate that 8-Bromo-cAMP attenuated IL-8 release by affecting signaling transductions induced by the particulate fraction.

Separation of transforming N-ras mutations in codons 12, 13 and 61 by denaturing gradient gel electrophoresis: investigation based on a set of phagemid constructs.

In the present study we have introduced 19 activating base pair substitutions into N-ras cDNA by use of an in vitro site-directed mutagenesis system. Six mutants were constructed for N-ras codon 12 (exon 1), six for codon 13 (exon 1), and seven for codon 61 (exon 2). Fifteen out of 19 PCR-amplified mutation sequences showed a clear separation from the wild type on denaturing gradient gel electrophoresis runs as homoduplex band, and the rest could be separated after heteroduplex formation with wild-type DNA. These constructs can be used as controls in many screening systems for analyzing activating point mutations of the N-ras gene.

Comparison of DGGE and CDGE in detection of single base changes in the hamster hprt and human N-ras genes.

Denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) are methods based on sequence-determined melting characteristics of DNA and thus detect different types of single base changes in the amplified fragments. We have studied detection of 19 mutations in the human N-ras oncogene and 10 mutations in exon 3 of the Chinese hamster hypoxanthine-guanine phosphoribosyltransferase (hprt) gene using GC-clamped DGGE and CDGE. After allowing formation of heteroduplexes with the corresponding wild type sequence, all the mutations separated from the wild type in at least one concentration of the denaturants used in CDGE but two of the mutations in hprt exon 3 did not show separation in any of the DGGE runs. Melting behavior of the mutant fragments was dependent, as expected, on both the type and the location of a mutation. We describe conditions allowing separation of the mutations in the fewest possible DGGE and CDGE runs.

A non-competitive chemiluminescence enzyme immunoassay for the equine acute phase protein serum amyloid A (SAA) -- a clinically useful inflammatory marker in the horse.

A non-competitive chemiluminescence enzyme immunoassay for measuring serum amyloid A (SAA) in equine serum was developed. A polyclonal anti-equine-amyloid A antiserum specific for equine SAA was utilized, and the assay was standardized using highly purified equine SAA. An acute phase horse serum was calibrated against the purified SAA and was used as standard when running the assay. Serum SAA concentrations in the range of 3-1210 mg/l could be measured. The reference range of SAA in clinically healthy adult horses was <7 mg/l. The clinical validation of the assay comprised the SAA responses after surgery and experimentally induced aseptic arthritis, and those associated with viral and bacterial infections. The SAA response after surgery (castration) was consistent, with peak concentrations on day 2 and a return to normal SAA concentrations within eight days. The aseptic arthritis produced an SAA response with a pattern similar to that seen after surgery, with peak concentrations of SAA 36-48 h after induction. Seven horses showed a biphasic pattern, with a second rise in SAA concentrations on day 4 and 5. All animals had SAA levels <7 mg/l on day 15. All horses with viral and bacterial infections had SAA concentrations above 7 mg/l. The ranges of SAA concentrations following the different types of inflammation overlap, being consistent with the unspecific nature of the SAA response. This study revealed that SAA is a sensitive and unspecific marker for inflammation, and describes the dynamics of the SAA response after standardized and well defined tissue damage.

Expression of TNFalpha and its receptors R1 and R2 in human alveolar epithelial cells exposed to organic dust and the effects of 8-bromo-cAMP and protein kinase A modulation.

OBJECTIVE: Expression of tumor necrosis factor alpha (TNF), TNF receptors 1 and 2 and TNFalpha converting enzyme (TACE) was studied in A549 human alveolar epithelial cells exposed to organic dust from a swine barn. Additional objectives were to elucidate whether 8-bromocAMP affected TNF and TNF receptor mRNA expression by activation of protein kinase A (PKA) and whether it increased phosphorylation of cAMP-responsive element-binding protein (CREB). MATERIALS AND METHODS: Reverse transcriptase- (RT-) PCR was performed on unexposed cells and cells exposed to a dust-suspension, with and without 8-bromo-cAMP (1 mM). H-89 was used to inhibit PKA. To further investigate mRNA expression of TNF, staurosporine was used. Immunolabeling was applied for detection of TNF, TNFR1, TNFR2 and phosphorylation of CREB. RESULTS: TNF mRNA and protein was expressed after 1-3 h in dust-exposed cells. TNFR2 mRNA and protein expression was induced by dust-exposure, whereas expression of TNFR1 and TACE was constitutive. After 1-1.5 h incubation, mRNA expression of TNF was (PKA-independently) attenuated by 8-bromo-cAMP (p < 0.05), whereas that of TNFR1 was PKA-dependently stimulated (p < 0.05). Staurosporine attenuated mRNA expression of TNF (p < 0.05), but not interleukin (IL)-6, which was detected prior to TNF. CONCLUSION: Expression of TNF and its receptors in alveolar epithelial cells may contribute to the response to organic dust. 8-bromo-cAMP, which increased the number of cells exhibiting phosphorylation of CREB exerted opposite effects on TNF and TNFR1 mRNA expression. The mechanism by which cAMP attenuates TNF mRNA expression remains to be established. Dust-induced expression of IL-6 precedes that of TNF and the induction pathways differ with regard to staurosporine sensitivity.

Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 the N-ras gene by constant denaturant capillary electrophoresis (CDCE).

We used high fidelity PCR and constant denaturant capillary electrophoresis (CDCE) [Khrapko et al. (1994) Nucleic Acids Res., 22, 364-369] to separate wild type and different mutant N-ras exon 1 and 2 sequences. The set of plasmids containing N-ras cDNA, wild type or mutant sequences representing all transforming amino acid-substituting single base pair changes in codons 12, 13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerase in a limited cycle polymerase chain reaction. One of the primers used for the amplification of each exon included a 40 nucleotide GC rich sequence that created high and low melting domains. The amplified fragments 151 bp (exon 1) and 150 bp (exon 2) were run on the CDCE with the 'denaturant zone' temperature of the capillary corresponding to the melting temperature of 111 bp (exon 1) and 110 bp (exon 2) low melting domains. The separation was achieved between wild type and mutant sequences as homoduplexes in 15 out of 19 cases, as a single base substitution alters the electrophoretic mobility of a partially melted double stranded fragment. The denaturation and reannealing of wild type and mutant fragments together created wild type/mutant heteroduplexes. All the heteroduplexes were well resolved from wild type homoduplex. In the current form mutant sequences were detected at a frequency of 10(-3) in the presence of wild type. This study has resulted in obtaining electrophoretic spectrum of different N-ras mutants on CDCE as homoduplexes as well as heteroduplexes.