Biomedical subjects

R Lucito

Publications and source records attributed to R Lucito.

7 recordsLinked to original sources

PubMed2000

Detecting gene copy number fluctuations in tumor cells by microarray analysis of genomic representations.

In this work, we explore the use of representations in conjunction with DNA microarray technology to measure gene copy number changes in cancer. We demonstrate that arrays of DNA probes derived from low-complexity representations can be used to detect amplifications, deletions, and polymorphic differences when hybridized to representations of genomic DNA. The method is both reproducible and verifiable, and is applicable even to microscopic amounts of primary tumors. We also present a mathematical model for array performance that is useful for designing and understanding DNA microarray hybridization protocols. The future applications and challenges of this approach are discussed.

Breast Neoplasms↗

PubMed1998

Genetic analysis using genomic representations.

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible "representations" of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

Blotting, Southern↗

PubMed1995

The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors.

The HBx protein of hepatitis B virus (HBV) is a transcriptional activator that is required for infection and may play an important role in HBV-associated hepatocarcinogenesis. Recently, we and others have shown that HBx stimulates the Ras-Raf-MAP kinase cascade, which leads to enhanced cell proliferation and the activation of transcription factors AP-1 and NF-kappa B. Other studies have shown that HBx can activate transcription by interacting directly with nuclear components of the transcription machinery. Therefore we examined the basis for the different reported activities of HBx. Here, we show that HBx is a complex protein, displaying independent activities in different intracellular locations. The intracellular distribution of HBx protein was first investigated using scanning confocal laser immunomicroscopy and by genetic studies. Our work has established that HBx expressed in cultured cells is found authentically in both the cytoplasm and the nucleus. HBx is not strongly associated with any intracellular structures, but some preferential accumulation was observed near the cell surface. Next, HBx variants were constructed containing a functional or mutant nuclear localization sequence. We show that when HBx is engineered to relocate exclusively to the nucleus, it no longer activates the Ras-Raf-MAP kinase cascade, nor does it activate transcription factors AP-1 and NF-kappa B. Surprisingly, nuclear HBx fully retains the ability to stimulate HBV enhancer I, which is activated independently of the Ras and protein kinase C pathways. Therefore HBx protein stimulates signal transduction pathways in the cytoplasm and transactivates transcription elements in the nucleus. Furthermore, SV40 T antigen is shown to induce the nuclear sequestration of HBx protein and to block its activation of NF-kappa B, demonstrating that HBx is regulated by proteins that alter its intracellular distribution. The conflicting functions of HBx protein in viral infection and possibly carcinoma may involve the regulation of its differential distribution in the cell.

Amino Acid Sequence↗

PubMed1992

Hepatitis B virus X protein activates transcription factor NF-kappa B without a requirement for protein kinase C.

The hepatitis B virus X protein stimulates transcription from a variety of promoter elements, including those activated by transcription factor NF-kappa B. A diverse group of extra- and intracellular agents, including growth factors and the human immunodeficiency virus tat protein, have been shown to require a functional protein kinase C (PKC) system to achieve activation of NF-kappa B. In this study we have investigated the molecular mechanism by which X protein activates NF-kappa B. We demonstrate that in hepatocytes, X protein induces a maximal activation of NF-kappa B corresponding to the sequestered pool of factor, which is also activated by phorbol esters. To determine whether X protein requires activation of PKC to stimulate transcription by NF-kappa B, we attempted to prevent transactivation by X protein in the presence of the PKC inhibitors calphostin C and H7. We show that PKC inhibitors do not block X protein activation of NF-kappa B, whereas they largely impair activation by phorbol esters. In addition, activation of PKC is correlated with its translocation from the cytoplasm to the plasma membrane. The subcellular distribution of PKC was investigated by introducing X protein from a replication-defective adenovirus vector, followed by immunochemical detection of PKC in cell fractions. These data also indicate that X protein stimulates transcription by NF-kappa B without the activation and translocation of PKC.

Carcinoma, Hepatocellular↗

PubMed1992

Alternate translation initiation on hepatitis B virus X mRNA produces multiple polypeptides that differentially transactivate class II and III promoters.

The hepatitis B virus X gene encodes a transcription activator which stimulates the synthesis of RNAs from a variety of class II and III promoter elements. In this report, we present a mutational analysis which genetically demonstrates that the X gene actually encodes two, and possibly three, related polypeptides from a single mRNA using alternate translation initiation from any of three in-frame AUG codons. Genetic analysis shows that translation initiates at the 5' proximal AUG of X mRNA and produces a full-length 17-kDa X protein but in addition also likely initiates at either of two conserved, in-frame AUG codons, producing two amino-terminally truncated X proteins presumably of 8 and 6.6 kDa. Expression of mRNAs capable of encoding only one of each X protein all individually transactivate class III (RNA polymerase III)-transcribed promoters. However, class II (RNA polymerase II)-transcribed promoters displayed various requirements for the different X proteins. Expression of two X proteins, the 17- and 6.6-kDa species, was required to activate transcription of the simian virus 40 enhancer/early promoter. In contrast, activation of an NF-kappa B-dependent promoter was carried out only by mRNAs encoding the full-length 17-kDa X protein. These results indicate that the X gene encodes several related proteins that possess different transcriptional regulatory activities.

Codon↗

PubMed1988

Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation.

Formation of the human U1 and U2 snRNA 3' ends requires both a conserved sequence, the 3' box, located downstream of the snRNA termini and sequences within the snRNA promoter regions. Indeed, replacement of the U1 snRNA promoter by mRNA promoters inhibits U1 3' end formation. We have now mutated the 5' flanking region of the human U2 gene and assayed the effects on initiation of transcription and 3' end formation. The 5' flanking region of the U2 gene contains two major promoter elements, a previously characterized distal element that enhances the efficiency of transcription and a proximal element, which our analysis localizes between positions -59 and -43 in a segment conserved in vertebrate snRNA genes. The 5' flanking region does not contain an element required solely for 3' end formation. However, when enhancer elements from an mRNA-encoding gene are introduced into a U2 promoter lacking its distal element, 3' end formation is inhibited. Together, these results suggest that the U2 promoter elements themselves are involved in 3' end formation, presumably by directing the formation of a unique transcription complex which is compatible with 3' end formation at the 3' box. Alteration of the composition of this transcription complex results in increased read-through at the 3' box.

DNA Mutational Analysis↗

PubMed1988

The effects of cortisone on acetylcholinesterase (AChE) in the neonatal and aged thymus.

Acetylcholinesterase (AChE) histochemistry and biochemistry was used to characterize the distribution and species of this enzyme within the developing thymus gland of the mouse. The results indicate that AChE-positive nerves and related structures are involved in a steroid-induced mechanism for regulating thymocyte populations. Low doses of cortisone injected into mice produce an activation of quiescent cholinergic nerves and the appearance of several new molecular forms of AChE within areas of the thymus where thymocyte death is prevalent. The action of cortisone on AChE is age dependent. In neonates, AChE activity is extremely high in the cortex of the gland, and cortisone causes little or no increase in AChE activity. In mice three to six weeks old, cortisone exerts its most profound effect on the AChE activity within the thymus. In mice eight months old and older, the AChE activity of the normal thymus is restricted to nerves and nerve-related structures at the cortical-medullary boundaries, with little or no activity observed in the cortex. Injections of cortisone in these mice does not cause an increase in AChE activity in the cortex and only slightly enhances activity within the cortico-medullary boundaries.

Acetylcholinesterase↗