Nonphysiological overexpression of low-density lipoprotein receptors causes pathological intracellular lipid accumulation and the formation of cholesterol and cholesteryl ester crystals in vitro.

Recent therapeutic strategies for the treatment of familial hypercholesterolemia have been based on liver-directed gene transfer of a functional low-density lipoprotein (LDL) receptor cDNA under control of viral or strong housekeeping promoters. Strong viral promoters including cytomegalovirus, Rous sarcoma virus, and simian virus 40 promoters are commonly employed to reach significant physiological effects. These promoters mediate constitutive and nonphysiological overexpression in every transduced cell, while the endogenous LDL receptor expression is controlled by a complex feedback mechanism based on intracellular cholesterol concentration. To investigate intracellular consequences of persistent LDL receptor overexpression we constructed a recombinant adenovirus encoding the human LDL receptor under control of the Rous sarcoma virus promoter. The metabolic and morphological effects of LDL receptor expression were characterized by uptake experiments with human hepatoma cells using fluorescent and radiolabeled LDL. We observed that large amounts of LDL accumulate within LDL receptor transduced cells, which eventually lead to massive intracellular lipid deposition. Kinetic experiments with LDL-supplemented medium resulted in numerous crystal shaped structures in the cytosol of transduced cells as visualized by digital interference contrast optic within 60 min after LDL supplementation. Thin layer chromatography analyses of cellular lipids suggested these crystalline structures to be dependent on intracellular cholesterol and cholesterol ester levels. Mock-infected cells showed neither cholesterol lipid accumulation nor crystal formation. In conclusion, our data demonstrate that nonphysiological overexpression of the LDL receptor can cause massive lipid accumulation, which cannot be compensated by the hepatoma cell metabolism. This phenomenon may result in negative selection of LDL receptor overexpressing cells in vitro and in vivo.

System for efficient helper-dependent minimal adenovirus construction and rescue.

Helper-dependent minimal adenoviral vectors deleted for all viral coding sequences are promising vectors for gene therapy. They retain only the adenovirus cis elements for replication and packaging, can accommodate up to 36 kb of foreign DNA, and exhibit prolonged transgene expression and reduced tissue toxicity as compared with first-generation adenoviral vectors. We have developed a system consisting of a set of cosmid cloning vectors (pMV and pMVX) for simple routine construction and efficient rescue of minimal adenoviral vectors. In the cloning vectors the inverted terminal repeats (ITRs) are flanked by recognition sites for the super rare-cutting endonuclease I-SceI. This allows the release of linear minimal adenovirus genomes for rescue of minimal adenovirus regardless of the sequence of the insert DNA. pMV contains a multiple cloning site for the insertion of 26 to 36 kb of therapeutic DNA. pMVX contains a noncoding human X-chromosomal DNA fragment as a vector backbone, which provides endonuclease restriction sites that allow for complete or partial replacement of the vector backbone by 1 to 26 kb of therapeutic DNA sequences, while retaining a packageable final minimal adenovirus genome size between 27 and 37.5 kb. Both vectors exist in two forms, with or without an Escherichia coli lacZ reporter gene cassette. Several minimal adenoviral vectors with insert sizes ranging from 1.5 to 16 kb were constructed with these cloning vectors. Minimal adenoviruses were efficiently rescued and amplified to high titers, using a Cre/lox-based helper system. Vectors containing the X-chromosomal backbone were stable during amplification. This simple and efficient system facilitates the construction of minimal adenoviruses and should be useful for further improvement of these new vectors.

Retinoids stimulate type I iodothyronine 5'-deiodinase activity in human follicular thyroid carcinoma cell lines.

Iodothyronine 5'-deiodinase isoenzymes generate the thyroid hormone 3,3',5-triiodothyronine from the prohormone L-T4. Basal and retinoic acid (RA)-induced type I 5'-deiodinase (5'DI) activities were studied in human thyroid carcinoma cell lines. In the follicular thyroid carcinoma line FTC-133, nanomolar concentrations of 9-cis, 13-cis-, and all-trans-RA induced 5'DI activity. Kinetics with all-trans-RA revealed 5'DI stimulation after 1 day and a maximal effect after 3 days. Increased abundance of the p27 5'DI subunit was demonstrated after RA treatment by N-bromoacetyl-[125I]T4 affinity labeling. Actinomycin-D and cycloheximide blocked RA-mediated induction. RA stimulated 5'DI activity to a lesser extent in FTC-238 cells, whereas neither basal 5'DI activity nor stimulation by RA was found in anaplastic thyroid carcinoma, human lung, or leukemia cell lines. Steady state messenger ribonucleic acid levels of RA receptor-alpha and -beta were increased after incubation of FTC-133 cells with all-trans-RA. The high 5'DI activity of differentiated rat thyroid FRTL-5 cells was not further induced by RA. Butyrate did not alter 5'DI, but increased the activity of the differentiation marker alkaline phosphatase in FTC-133 and FTC-238 cells. T4 and T3 had no effect on basal or RA-stimulated 5'DI activity. These data suggest that expression and retinoid induction of 5'DI may serve as a sensitive and functional differentiation parameter of follicular thyroid carcinoma cells.

A bovine homologue of the human TSPY gene.

We have isolated a male-specific bovine genomic homologue of the human TSPY gene (testis-specific protein, Y-encoded). Two specific transcripts can be detected in bovine testicular RNA. The cloned sequence represents one copy of a clustered array of some 50-200 related sequences on the Y chromosome resembling the arrangement present in the human genome. The clone isolated from the bovine genome has two regions that are highly conserved between homo and bos. One stretch of DNA is homologous in 45 of 51 bp (88%), and the other stretch shares 71 of 100 bp (71%).

TSPY-related sequences represent a microheterogeneous gene family organized as constitutive elements in DYZ5 tandem repeat units on the human Y chromosome.

TSPY (testis-specific protein, Y-encoded) is encoded by members of a Y-chromosome-specific sequence family. We show here that TSPY elements are part of the DYZ5 repeat unit. We have established a cosmid library of Y-chromosomal DNA derived from the hybrid cell line 3E7 and isolated eight cosmids representing 15 TSPY elements. Interindividual variability with respect to TSPY copy number was observed. One cosmid clone was investigated in detail by subcloning and sequencing. Sequence microheterogeneity was identified among both transcribed and nontranscribed TSPY elements. Transcript variability is not restricted to single basepair exchanges. We present data for the existence of at least three differently sized TSPY transcripts, caused by different patterns of splicing.

Testis-specific protein, Y-encoded (TSPY) expression in testicular tissues.

TSPY, the 'testis-specific protein, Y-encoded', is the product of a tandem gene cluster on human proximal Yp. In order to gain insight into the function of this locus, we have analysed (I) the diversity of RNAs transcribed from the cluster, (II) the sequence homology of the deduced TSPY to other proteins, and (iii) its protein properties both in tissue extracts and in tissue sections, using a TSPY-specific antiserum. We have identified a set of distinct TSPY transcripts with diverse exon compositions. We show that TSPY has homology with other human and non-human proteins, including SET and NAP, factors that are suggested to play a role in DNA replication. Protein analysis revealed TSPY to occur mainly in a modified, putatively phosphorylated form. By immunostaining it was detected in distinct subsets of spermatogonia. TSPY was also strongly immunostained in early testicular carcinoma in situ (CIS), while seminomatous tumour cells stained less intensely. The spermatogonial cells of two XY-TFM-females gave a strong immune response. The data presented here point to a phosphorylation-dependent TSPY-function in early spermatogenesis, immediately prior to the spermatogonia-to-spermatocyte transition, and in early testicular tumorigenesis.

Murine and human TSPYL genes: novel members of the TSPY-SET-NAP1L1 family.

Using expressed sequence tag (EST) sequence information, novel genes related to the Y chromosome gene family TSPY were isolated and characterized. Because of a significant homology to TSPY, the novel genes were designated TSPY-Like (TSPYL) and were assigned as new members of the TSPY-SET-NAP1L1 family. A human TSPYL gene was localized on chromosome 6 and a murine Tspyl gene was localized on chromosome 10. Expression of TSPYL was observed in all tissues investigated, as well as early onset during development. Both the human and murine Tspyl homologs lack introns over the entire region so far investigated and are thought to have arisen by an ancient retroposition event. Retroposition of Tspyl genes is supported by the isolation of a murine Tspyl pseudogene on chromosome 12 which also lacks intronic sequences, and by its observed proximity to an R element, a family of dispersed repetitive DNA.

A new missense substitution at a mutational hot spot of the androgen receptor in siblings with complete androgen insensitivity syndrome.

Several mutations have been described in the human androgen receptor gene including constitutional mutations in androgen insensitivity syndrome, somatic mutations in prostate cancer and triplet expansions in Kennedy's disease (Gottlieb et al. 1997). Here we report on two siblings with complete androgen insensitivity and a novel missense mutation, D695V, in their androgen receptor gene. The two XY females are siblings of German descent and presented at the ages of 23 and 19 years, respectively, with typical clinical features of complete androgen insensitivity. We found both siblings to be hemizygous for a new adenine to thymine transversion at the second nucleotide of codon 695 within the fourth exon of the human androgen receptor gene. The resulting missense mutation D695V is located at the amino-terminal border of the ligand-binding domain of the androgen receptor. The aspartic acid residue at this position is highly conserved in the steroid binding domains of other members of the nuclear receptor family and has already been found to be the site of two other missense mutations associated with androgen insensitivity syndrome (Ris Stalpers et al. 1991, Hiort et al. 1996). Three of four reported subjects showed the complete androgen insensitivity phenotype, in accordance with the two siblings in our study. We suggest that the existence of three pathological amino acid substitutions for aspartic acid 695 most likely reflects the essential role of this residue for normal androgen receptor function in male sexual differentiation.

Organization and expression of bovine TSPY.

We have isolated genomic sequences as well as transcripts from the bovine homolog of the human testis-specific protein, Y-encoded, TSPY which-in both species-is located on the Y Chromosome (Chr), organized as a gene family with a variable number of members, and expressed exclusively in the testis. 1266 bp of bovine TSPY specific sequence have been isolated from a testis cDNA library, by RT-PCR analyses and by Rapid Amplification of cDNA Ends (RACE). A bovine TSPY gene 4 is organized in seven exons, and transcripts are polyadenylated at various 3' ends. Consensus polyadenylation signals AAUUAAA are missing. Microheterogeneous sequence variation is found between TSPY family members. In addition, homologies to other Y-located repeated sequence families, BRY, have been discovered; these sequences are presumably derived from ancient members of the TSPY cluster, now forming a separate, probably nonfunctional subfamily. Bovine TSPY is subject to differential splicing. In the adult, it is expressed in early germ-cell stages, and expression could also be detected in fetal testis. Comparison with the human homolog shows the highest degree of similarity in the coding regions of exons 2, 3, and 4, which are also precisely conserved regarding their length.

Intravenous administration of recombinant adenoviruses causes thrombocytopenia, anemia and erythroblastosis in rabbits.

BACKGROUND: Recombinant adenoviruses are highly efficient gene transfer vehicles but their administration to mammals is accompanied by a strong inflammatory response. The present study reports additional side effects observed during adenoviral gene transfer studies in rabbits. METHODS: Hematological and serological parameters, the course of viremia and the organ distribution were analyzed after in vivo administration of E1-deleted adenoviruses in rabbits. RESULTS: The systemic administration of a therapeutic dose of 5 x 10(11) infectious particles/kg (infusion time 20 min) led to an average reduction of 80-90% in the platelet count within 48 h. Full recovery took 10-14 days. Virus administration induced a strong but transient erythroblastosis (peaking 24 h after administration) which settled 48 h later. Normochromic anemia occurred over the next 10 days with hemoglobin levels dropping by about 40% to reach the lowest level 10 days after administration and taking two months for full recovery. Dose-dependent thrombocytopenia was also found in mice, but neither erythroblastosis nor anemia was observed (in equivalent doses). The hematological findings did not improve after local injection via the portal vein. Local and systemic administration led to a comparable course of viremia. Only minor differences were found in the biodistribution of viruses between local and systemic administration. Large amounts of viral DNA and transgene expression were found in the lungs, the kidneys and the ovaries, even after local administration via the portal vein. CONCLUSIONS: Local intravenous injection via the portal vein does not prevent systemic spread of viral vectors and the occurrence of vector-related side effects. The hematological changes observed in rabbits suggest the need for careful monitoring of hematological and rheological parameters in clinical trials.