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Daniel T Starczynowski

Publications and source records attributed to Daniel T Starczynowski.

6 recordsLinked to original sources
PubMed2005

Mutations of tumor necrosis factor alpha-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability.

The human c-rel gene (REL), encoding an NF-kappaB transcription factor, is amplified or mutated in several human B-cell lymphomas and can transform chicken lymphoid cells in vitro. We have previously shown that certain deletions of C-terminal transactivation sequences enhance REL's transforming ability in chicken spleen cells. In this report, we have analysed the effect of single amino-acid changes at select serine residues in the C-terminal transactivation domain on REL's transforming ability. Mutation of either of two TNFalpha-inducible serine residues (Ser460 and Ser471) to nonphosphorylatable residues (alanine, asparagine, phenylalanine) made REL more efficient at transforming chicken spleen cells in vitro. In contrast, mutation of Ser471 to a phosphorylation mimetic aspartate residue impaired REL's transforming ability, even though it increased REL's inherent transactivation ability as a GAL4-fusion protein. Alanine mutations of several other serine residues within the transactivation domain did not substantially affect REL's transforming ability. Transactivation by GAL4-REL fusion proteins containing either transformation enhancing or nonenhancing mutations at serine residues was generally similar to wild-type GAL4-REL. However, more transforming mutants with mutations at either Ser460 or Ser471 differed from wild-type REL in their ability to transactivate certain kappaB-site reporter genes. In particular, the SOD2 promoter, encoding manganese superoxide dismutase, was activated less strongly by the more transforming REL mutant REL-S471N in transient assays, but REL-S471N-transformed chicken spleen cells had increased levels of MnSOD protein as compared to wild-type REL-transformed cells. Taken together, our results show that mutations of certain serine residues can enhance REL's transforming ability in vitro and suggest that these mutations increase REL-mediated transformation by altering REL's ability to modulate the expression of select target genes. Furthermore, phosphorylation of Ser471 may be involved in REL-mediated modulation of transformation-specific target gene expression. Lastly, these results suggest that similar mutations in the REL transactivation domain contribute to the development of certain human B-cell lymphomas.

Animals↗
PubMed2004

Characterization of a human REL-estrogen receptor fusion protein with a reverse conditional transforming activity in chicken spleen cells.

Overexpression of the human REL transcription factor can malignantly transform chicken spleen cells in vitro. In this report, we have created and characterized a cDNA encoding a chimeric protein (RELDelta424-490-ER) in which sequences of a highly transforming REL mutant (RELDelta424-490) are fused to the ligand-binding domain of the human estrogen receptor (ER). Surprisingly, RELDelta424-490-ER is constitutively nuclear in A293 cells, and RELDelta424-490-ER activates transcription in the absence, but not in the presence, of estrogen in kappaB-site reporter gene assays. Furthermore, RELDelta424-490-ER transforms chicken spleen cells in the absence of estrogen, but the addition of estrogen blocks the ability of RELDelta424-490-ER-transformed cells to form colonies in soft agar, even though estrogen induces increased nuclear translocation of RELDelta424-490-ER in these cells. ERalpha can also inhibit REL-dependent transactivation in trans in an estrogen-dependent manner, and ERalpha can interact with REL in vitro. Thus, the RELDelta424-490-ER fusion protein shows an unusual, reverse hormone regulation, in that its most prominent biological activities (transformation and transactivation) are inhibited by estrogen, probably due to an estrogen-induced interaction between the ER sequences and sequences in the Rel homology domain. Nevertheless, these results indicate that the continual activity of REL is required to sustain the transformed state of chicken spleen cells in culture, suggesting that direct and specific inhibitors of REL may have therapeutic efficacy in certain human lymphoid cancers.

Animals↗
PubMed2004

The c-Rel transcription factor and B-cell proliferation: a deal with the devil.

Activation of the Rel/NF-kappaB signal transduction pathway has been associated with a variety of animal and human malignancies. However, among the Rel/NF-kappaB family members, only c-Rel has been consistently shown to be able to malignantly transform cells in culture. In addition, c-rel has been activated by a retroviral promoter insertion in an avian B-cell lymphoma, and amplifications of REL (human c-rel) are frequently seen in Hodgkin's lymphomas and diffuse large B-cell lymphomas, and in some follicular and mediastinal B-cell lymphomas. Phenotypic analysis of c-rel knockout mice demonstrates that c-Rel has a normal role in B-cell proliferation and survival; moreover, c-Rel nuclear activity is required for B-cell development. Few mammalian model systems are available to study the role of c-Rel in oncogenesis, and it is still not clear what features of c-Rel endow it with its unique oncogenic activity among the Rel/NF-kappaB family. In any event, REL may provide an appropriate therapeutic target for certain human lymphoid cell malignancies.

Animals↗
PubMed2003

Deletion of either C-terminal transactivation subdomain enhances the in vitro transforming activity of human transcription factor REL in chicken spleen cells.

The REL gene is amplified in many human B-cell lymphomas and we have previously shown that expression of REL from a retroviral vector can malignantly transform chicken spleen cells in vitro. To identify REL protein functions necessary for malignant transformation, we have performed deletion analysis on REL sequences encoding residues of two C-terminal subdomains that are involved in transcriptional activation. We find that deletion of both C-terminal transactivation subdomains abolishes the ability of REL to transform chicken spleen cells in vitro. In contrast, deletion of either transactivation subdomain alone, which reduces the transactivation ability of REL, enhances the transforming activity of REL. Transforming REL mutants missing C-terminal sequences can also be selected at a low frequency in vitro. The REL transactivation domain can be functionally replaced in transformation assays by a portion of the VP16 transactivation domain that activates at a level similar to REL-transforming mutants. We also find that deletion of 29 C-terminal amino acids causes the subcellular localization of REL to change from cytoplasmic to nuclear in chicken embryo fibroblasts. In contrast, wild-type REL and all transforming REL mutants are located primarily in the cytoplasm of transformed spleen cells. Nevertheless, treatment of transformed spleen cells with leptomycin B causes wild-type REL and two REL mutants to relocalize to the nucleus, and nuclear extracts from these transformed cells contain REL DNA-binding activity. Taken together, these results suggest the following: (1) that REL must activate transcription to transform cells in vitro; (2) that a reduced level of transactivation enhances the oncogenicity of REL; (3) that REL shuttles from the cytoplasm to the nucleus in transformed chicken spleen cells; and (4) that mutations in REL, in addition to amplifications, could activate its oncogenicity in human lymphomas.

Amino Acid Sequence↗
PubMed2003

Polymeric FAD used as enzyme-friendly mediator in lactate detection.

We present the fabrication and properties of lactate biosensors. The novel feature is the use of polymerized flavin adenine dinucleotide (FAD) as mediator for electron transfer. The biosensors were prepared using lactate dehydrogenase (LDH), lactate oxidase (LOX), or baker's yeast (BY) immobilized at the surface of the electrode. The sensors using purified enzymes showed good sensitivity, linearity, and stability. The sensitivity of the BY electrodes was slightly lower. The advantages of this type of sensors are discussed in connection with potential applications.

Biopolymers↗