Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34-36, 50-60, 62-68, 77-78, 104-110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01-25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by approximately 20-50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.
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PMID:Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction. 129 28

5-HT1c receptors have been shown to act as protooncogenes in NIH 3T3 cells, inducing ligand-dependent focus formation. In order to assess their mitogenic and oncogenic potential in a different cell system, we transfected these receptors into CCL39 hamster fibroblasts, a well-characterized growth factor-dependent cell line. Cell clones expressing functional receptors were isolated and tested for (a) growth factor dependence of proliferation measuring thymidine incorporation in response to varying doses of serum, (b) the response to serotonin alone or in combination with other growth factors, and (c) the capacity for anchorage-independent proliferation. In the absence or presence of serotonin, the large majority of the clones isolated showed normal morphology and normal growth factor dependence and was unable to grow in soft agar. None of the clones showed a significant response to serotonin alone in DNA synthesis reinitiation experiments, but synergy was observed between serotonin and the tyrosine kinase activating growth factors EGF and FGF. However, the major part of this effect could be abolished by an antagonist of 5-HT1b receptors, which are endogenous in CCL39 cells. The same receptor was found to mediate a significant mitogenic response to the neurotransmitter in Ha-ras-transfected cells. The fact that 5-HT1c receptors do not readily induce a transformed phenotype in CCL39 cells clearly distinguishes them from strong dominantly acting oncogene products like RAS, SRC, or FMS.
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PMID:Effects of 5-HT1C-receptor expression on cell proliferation control in hamster fibroblasts: serotonin fails to induce a transformed phenotype. 131 91

The polymerase chain reaction (PCR) cannot be used to amplify the breakpoint in the chimaeric BCR-ABL gene in CML and acute leukaemias due to the large variation in the sites of breakpoint in the BCR gene (within a 5.8 kb region) and in the ABL gene (within a 150 kb region). The disease state is usually monitored using RNA-PCR to monitor abnormal transcripts. We have used a new modification of the PCR to amplify breakpoints within zone 3 of the M-bcr. A synthetic oligonucleotide linker, the Vectorette, is ligated to restriction digested DNA, and amplification is carried out between primers for a known target sequence and the Vectorette linker. Three Philadelphia chromosome Ph1-positive CML patients with breakpoints within the ALU region of zone 3 have been amplified and the sequence immediately around the breakpoint determined. The breaks occurred within 70 bp and two were only 14 bp apart. The Vectorette-PCR technique has the potential to rapidly identify and sequence breakpoints, and will enable the design of patient-specific primers to monitor disease progression, particularly following bone marrow transplantation.
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PMID:Amplification and sequencing of genomic breakpoints located within the M-bcr region by Vectorette-mediated polymerase chain reaction. 131 90

We have compared the efficacy of digoxigenin- and biotin-labelled probes in detecting HPV DNA by in situ hybridization on paraffin-embedded tissue sections of 57 male condyloma-suspect genital lesions. Each biopsy was hybridized with at least three of the following four methods: digoxigenin-labelled HPV DNA probes (Dig-HPV), biotinylated HPV-DNA probes (Bio-HPV), and two commercial methods (ViraType in situ and PathoGene), both based on biotinylated DNA probes. The hybridization products were visualized with colourigenic enzyme substrates. In most biopsies, the 4 methods gave equal results although cross-hybridization was most often found with the low-stringency ViraType method. Dig-HPV 6/11 probes gave positive results about twice as often as either of the commercial methods. No such difference, however, was found for HPV 16/18 probes. DNA of any type of HPV 6/11, 16/18 or 31/33/35 or 51 was detected in 28/43 (65%) of lesions showing condyloma acuminatum histology but in none of the 14 biopsies with no histological signs of HPV infection. In HPV-positive condylomata with no cellular atypia. HPV 6/11 was detected in 87% (13/15), and HPV 16/18 in 27% (4/15). In biopsies with cellular atypia, HPV types 6/11 were detected in 62% (8/13), HPV types 16/18 in 46% (6/13), and HPV types 31/33/35 or 51 in 50% (6/12). In about 50% of the biopsies where at least one hybridization method gave a positive result, either one of the commercial methods gave a negative result.(ABSTRACT TRUNCATED AT 250 WORDS)
Int J STD AIDS
PMID:Comparison of four in situ hybridization methods, based on digoxigenin- and biotin-labelled probes, in detecting HPV DNA in male condylomata acuminata. 131 47

A series of 65 male sexual partners of 65 women attending an STD clinic in Bologna, Italy for examination and treatment of genital human papillomavirus (HPV)-infections during 1990-1991, were examined using peniscopy and surgical biopsy, the latter being analysed by light microscopy, in situ hybridization (ISH) and polymerase chain reaction (PCR) for HPV DNA. A detailed medical and sexual history was recorded from all men. Of the 65 men, 17 (26.2%) gave a history of a previous STD. The male partners with previous genital condylomata (14, 21.5% of men) were significantly associated with the detection of HPV DNA in the current lesions; 21.4% (3 of 14) and 10.2% (5 of 51) in those with and without previously treated condyloma, respectively. On colposcopy, 63 (96.9%) men presented with an abnormal pattern, the vast majority (49 of 65, 75.4%) showing an acetowhite lesion, and only 12 (18.5%) lesions being classified as condyloma acuminatum. HPV DNA was found, however, in only 4 of 12 (33.3%) condylomas by ISH and PCR, and in 4 of 49 (8.2%) and 6 of 49 (12.2%) acetowhite lesions by ISH and PCR, respectively. In a total of 41 (63%) patients, the biopsy was classified as non-HPV on light microscopy. HPV DNA detection rate was significantly higher in all morphologically HPV-suggestive lesions, compared with the non-HPV where ISH was invariably negative. PCR, however, disclosed HPV DNA in 4 of 41 (9.8%) cases. PIN (I or II) was present in 6 of 65 (9.2%) men.(ABSTRACT TRUNCATED AT 250 WORDS)
Int J STD AIDS
PMID:Detection of human papillomavirus infections in the male sexual partners of women attending an STD clinic in Bologna. 132 74

The involvement of the BCRlABL fusion gene in patients with Philadelphia (Ph) chromosome positive chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL) is well characterised, but the molecular events underlying the cases of Ph-negative CML and ALL that lack BCR gene involvement and those that cause transformation of Ph-positive CML are unknown. The murine ABL gene can be activated by genetic events that do not involve the BCR gene, including the introduction of two specific point mutations in exons VII and XI respectively, as found in the homologous sequence of the v-abl oncogene. We therefore sought evidence for analogous point mutations in the ABL gene in patients with Ph-negative, BCR-negative CML (n = 25), Ph-negative ALL (n = 18) and in Ph-positive CML in transformation (n = 28). We used restriction fragment length polymorphism and single strand conformational polymorphism techniques to analyse DNA amplified fragments of selected ABL coding regions from leukaemia cells. We identified only normal wild-type DNA sequences. The absence of these transforming point mutations does not exclude the possibility that the ABL gene in such patients could be activated by other means.
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PMID:Specific point mutations that activate v-abl are not found in Philadelphia-negative chronic myeloid leukaemia, Philadelphia-negative acute lymphoblastic leukaemia or blast transformation of chronic myeloid leukaemia. 135 50

Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.
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PMID:Differential expression of the mouse and human Thy-1 gene in embryonal carcinoma cells. 136 23

We performed molecular studies to resolve the status of BCR and ABL in the bone marrow cells of a CML patient with a Ph chromosome resulting from a complex translocation involving chromosomes 9, 15, and 22. DNA digestion with BamHI, HindIII, and BglII, followed by hybridization to a bcr-specific 32P-labeled probe, showed a rearranged banding pattern confirming the involvement of the bcr locus in the translocation. Furthermore, total cellular RNA isolated from the marrow was subjected to reverse transcription into cDNA and amplified by PCR with primers specific for BCR-ABL fusion cDNA. The amplified products obtained from this patient and from a CML patient with the standard t(9;22) were both of the expected length of approximately 317 bp.
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PMID:Molecular confirmation of BCR-ABL fusion in a chronic myeloid leukemia with a complex translocation involving chromosomes 9, 15, and 22. 137 43

Preliminary studies of RAS mutational activation in human testicular germ cell neoplasms have yielded conflicting results. Whereas two studies of clinical material revealed a significant incidence of N- and KRAS mutations, two studies of a variety of germ cell lines failed to document RAS mutations. To clarify the incidence of RAS mutations in these tumors, we studied archival paraffin-embedded, formalin-fixed orchiectomy specimens from 25 nonseminomas (NSGCT), 18 seminomas (SEM), and one Leydig cell tumor. For 14 of the 44 neoplasms, DNA was also available from nonmalignant testis adjacent to the tumor. Six age-matched patients had testes removed because of nonmalignant disease and were studied as controls. Polymerase chain reaction (PCR) amplified the K-, N-, and HRAS 12, 13, and 61 codons of these specimens, and mutations were detected with mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Four mutations were found in KRAS 12 (4/44;[9.1%]). One seminoma [1/18(5.6%)] contained the mutation GGT(GLY)----CGT(ARG), and three NSGCT [3/25(12%)] were found to have GGT(GLY)----GAT(ASP) mutations. One of the NSGCT mutations was detected in adjacent nonmalignant tissue, but the corresponding tumor did not contain any detectable mutation. No mutations were detected at KRAS 13 or 61, in NRAS or HRAS 12, 13, or 61, or in the control normal testes. PCR, slot blots, and hybridizations were performed twice by two separate investigators for confirmation of results. PCR-generated mutation-specific positive controls were created for all possible RAS mutations, and these along with wild-type DNA controls were integral to interpretation of the oligonucleotide mismatch hybridization assay. By using positive and negative controls, we have detected a relatively low incidence of RAS mutations in archival human testicular germ cell tumors.
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PMID:Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization. 138 46

Advances in molecular genetics in the past decade enabled us to analyze the cause of mendelian disorders at molecular level and a variety of mutations, not only in point mutations and deletion in exons but also in those occurred in regulatory elements or in RNA processing have been precisely identified. Such a variety of mutations may constitute variable clinical manifestations even in the simple mendelian disorders. On the other hand, pathogenesis of common diseases is much complicated and remains greatly to be elucidated. However, if we could use the strategies applied in the past few years for mendelian disorders, it seems to be not difficult to approach them. It is recommended to categorize a certain disease into subgroups for distinguishing their heterogenous phenotypes by clinical, biochemical and other properties. Owing to the success in making a subgroup (FAB classification), many subtype-specific translocations were found in leukemia, and then, rearrangement of relevant genes is also being shown. The best example is seen in chronic myelocytic leukemia. Since rearrangement of ABL and BCR was shown and both genes were cloned, detection of minimal residual diseases after intensive treatment became possible at 10(-6) level using RT-PCR technique. Recently developed interphase cytogenetics using FISH has visualized Ph1 translocation in metaphase cells and also in round nuclei, suggesting a potential use in monitoring the effect of certain drugs during treatment. Furthermore, very selective targeting therapy is being devised using antisense DNA.
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PMID:[Present status of gene diagnosis in cancer]. 144 79


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