DSC3 expression is regulated by p53, and methylation of DSC3 DNA is a prognostic marker in human colorectal cancer.

BACKGROUND: Desmocollin 3 (DSC3), a member of the cadherin superfamily and integral component of desmosomes, is involved in carcinogenesis. However, the role of DSC3 in colorectal cancer (CRC) has not yet been established. METHODS: Desmocollin 3 expression in CRC cell lines was analysed by RT-PCR and western blotting. Methylation status of DSC3 was examined by demethylation tests, methylation-specific PCR, and bisulphite sequencing (BS). The regulatory role of p53 was investigated by transfection. RESULTS: Desmocollin 3 was downregulated in CRC cells at mRNA and protein levels. Desmocollin 3 expression was restored in five out of seven cell lines after 5-aza-2'-deoxycytidine (DAC) treatment. A heterogeneous methylation pattern was detected by BS in promoter region and exon 1 of DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out of 99) of primary CRC, being associated with poor prognosis (P=0.002). Transfection of p53 alone or in combination of DAC increased the DSC3 expression. Similarly, treatment with p53 inducer adriamycin alone or in combination with DAC enhanced DSC3 expression. CONCLUSIONS: DNA methylation contributes to downregulation of DSC3 in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker for CRC. P53 appears to have an important role in regulating DSC3 expression.

Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector.

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.

Regulation of the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 3.

pp72syk is essential for development and function of several hematopoietic cells, and it becomes activated through tandem SH2 interaction with ITAM motifs in immune response receptors. Since Syk is also activated through integrins, which do not contain ITAMs, a CHO cell model system was used to study Syk activation by the platelet integrin, alpha IIb beta 3. As in platelets, Syk underwent tyrosine phosphorylation and activation during CHO cell adhesion to alpha IIb beta 3 ligands, including fibrinogen. This involved Syk autophosphorylation and the tyrosine kinase activity of Src, and it exhibited two novel features. Firstly, unlike alpha IIb beta 3-mediated activation of pp125FAK, Syk activation could be triggered by the binding of soluble fibrinogen and abolished by truncation of the alpha IIb or beta 3 cytoplasmic tail, and it was resistant to inhibition by cytochalasin D. Secondly, it did not require phosphorylated ITAMs since it was unaffected by disruption of an ITAM-interaction motif in the SH2(C) domain of Syk or by simultaneous overexpression of the tandem SH2 domains. These studies demonstrate that Syk is a proximal component in alpha IIb beta 3 signaling and is regulated as a consequence of intimate functional relationships with the alpha IIb beta 3 cytoplasmic tails and with Src or a closely related kinase. Furthermore, there are fundamental differences in the activation of Syk by alpha IIb beta 3 and immune response receptors, suggesting a unique role for integrins in Syk function.

Association of newly discovered IS elements with the dichloromethane utilization genes of methylotrophic bacteria.

Dichloromethane (DCM) dehalogenases enable facultative methylotrophic bacteria to utilize DCM as sole carbon and energy source. DCM-degrading aerobic methylotrophic bacteria expressing a type A DCM dehalogenase were previously shown to share a conserved 4.2 kb BamHI DNA fragment containing the dehalogenase structural gene, dcmA, and dcmR, the gene encoding a putative regulatory protein. Sequence analysis of a 10 kb DNA fragment including this region led to the identification of three types of insertion sequences identified as IS1354, IS1355 and IS1357, and also two ORFs, orf353 and orf192, of unknown function. Two identical copies of element IS1354 flank the conserved 4.2 kb fragment as a direct repeat. The occurrence of these newly identified IS elements was shown to be limited to DCM-utilizing methylotrophs containing a type A DCM dehalogenase. The organization of the corresponding dcm regions in 12 DCM-utilizing strains was examined by hybridization analysis using IS-specific probes. Six different groups could be defined on the basis of the occurrence, position and copy number of IS sequences. All groups shared a conserved 5.6 kb core region with dcmA, dcmR, orf353 and orf192 as well as IS1357. One group of strains including Pseudomonas sp. DM1 contained two copies of this conserved core region. The high degree of sequence conservation observed within the genomic region responsible for DCM utilization and the occurrence of clusters of insertion sequences in the vicinity of the dcm genes suggest that a transposon is involved in the horizontal transfer of the DCM-utilization character among methylotrophic bacteria.

Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction.

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.

Differential transfection efficiency rates of the GM-CSF gene into human renal cell carcinoma lines by lipofection.

One of the major questions in any gene therapy approach is the selection of the appropriate vector system. Here, the optimization of a gene transfer protocol for renal cell carcinoma using lipofection as a nonviral gene transduction system was evaluated. To select the promoter which gives the highest expression, different plasmids which are able to express Escherichia coli beta-galactosidase gene as a reporter gene under the control of different promoters were tested: human cytomegalovirus promoter (pCMVbeta), simian virus 40 promoter (pSVbeta), adenovirus promoter (ADbeta), and herpes simplex virus thymidine kinase promoter (TKbeta). The pCMVbeta revealed the highest expression of the beta-gal gene in the renal cell carcinoma (RCC) lines. Thus this CMV promoter was selected for the expression of the granulocyte-macrophage colony stimulator factor (GM-CSF) gene. Three different lipids (LipofectAmine, LipofectAce, and Lipofectin) were compared for their transduction efficiency, and the optimal conditions for quantitatively high lipofection rates were established. The consistently best results regarding gene expression as well as viability of the RCC lines were obtained when Lipofectin was used. Gene expression was monitored by a specific enzyme-linked immunosorbent assay and functionally validated by a cell proliferation test. The GM-CSF expression profile showed a peak at 48 hours after transfection and was still detectable after 5 days. Here the feasibility of efficient lipofection of the GM-CSF gene into RCC lines is demonstrated. Most importantly, considerable differences in the relative quantity of GM-CSF gene transfer into the different RCC lines was observed here. This may be of critical relevance for the design of any clinical gene transduction protocol in tumor cell vaccination attempts.

Cessation of immunosuppressive therapy after successful transplantation: a national survey.

A retrospective survey of 165 United States renal transplant units identified 48 patients who had discontinued all immunosuppressive therapy (IT). Sixteen had received cadaveric and 32 had received living-related grafts. The interval from transplantation to cessation of medication was similar in both groups and did not correlate with the outcome. Of 16 cadaveric grafts, 9 failed ("delta" serum creatinine concentration of greater than or equal to 5 mg/dl) within a mean of 59 days; 3 patients maintained stable renal function for 244, 395, and 425 days, respectively. Of the 32 related grafts, 12 failed within a mean of 234 days. Of the 20 related grafts that did not fail, 5 had stable function for greater than or equal to 1 year and 6 for greater than or equal to 3 years after cessation of IT. Our data demonstrate that at no point after transplantation is it prudent to stop all IT barring serious drug toxicity. Also, in patients who stop IT surreptitiously and in whom renal function remains normal, reinstitution of therapy is indicated within 1 year and is advisable as long as 3 years after cessation; those few patients who do well without IT for greater than 3 years may not need further treatment.