Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin

Summary The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficacious system for somatic cell gene therapy. It is likely that any ex vivo system will require the use of large numbers of cells which express high levels of transgene products. We present a method for routine expansion of canine bone marrow stromal cells, established from initial 10–20 ml marrow aspirates, to greater than 10⁹ cells. This high level expansion of cell cultures uses the stimulatory effect of acidic fibroblast growth factor (aFGF) and heparin. In the absence of these factors, stromal cell cultures grow actively for only 1 to 2 passages, become flattened in morphology, and expand to only 10⁸ cells. In the presence of heparin (5 U/ml), aFGF exerts its effect over a wide range of concentrations (0.1–10 ng/ml) in a dose-dependent manner. The stimulatory effect is dependent on the presence of both aFGF and heparin. Immunocytochemical and cytochemical analyses phenotypically characterize these stromal cells as bone marrow stromal myofibroblasts. Stromal cells grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages, transduce efficiently with a human growth hormone (hGH) expression vector, and express and secrete high levels of hGH. Human marrow stromal cells were also established and expanded by the same culture method. This culture method should be of great value in somatic cell gene therapy for the delivery of secreted gene products to the plasma of large mammals.     

Molecular analysis of the acid sphingomyelinase deficiency in a family with an intermediate form of Niemann-Pick disease.

A novel point mutation in the lysosomal acid sphingomyelinase gene has been identified in the recently reported Serbian family with a clinically and biochemically atypical intermediate form of Niemann-Pick disease. The mutation was a T1171-->G transversion resulting in substitution of glycine for normal tryptophan at amino acid residue 391. The coding sequence was otherwise normal. All of the five affected individuals were almost certainly homoallelic, and both of the two obligate heterozygotes studied also carried the same mutation. This mutation is therefore likely to be directly associated with the atypical phenotype of these patients. Expression in COS-1 cells suggested a higher residual activity than that in cultured fibroblasts. A recently developed high-affinity rabbit antihuman sphingomyelinase antibody allowed us to study for the first time the biosynthesis, processing, and targeting of a mutant sphingomyelinase by metabolic labeling of cultured fibroblasts. The mutant enzyme protein was normally synthesized, processed, and routed to the lysosome but was apparently unstable and degraded rapidly once it reached the lysosome. Together with the finding of the relatively high residual activity in COS-1 cells, we interpret our observations to mean that instability and rapid breakdown of the mature mutant enzyme protein, due to the mutation rather than direct inactivation of the catalytic activity, is the primary mechanism for the deficiency of sphingomyelinase activity in these patients. A high prevalence of this mutation in the Serbian population is likely, since the family pedigree indicates that members from four reportedly unrelated families must have contributed the same mutation.     

Human RPA (hSSB) interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus.

RPA is the replicative single-strand DNA (ssDNA) binding protein of eukaryotic chromosomes. This report shows that human RPA interacts with EBNA1, the latent origin binding protein of Epstein-Barr virus (EBV). RPA binds to EBNA1 both in solution, and when EBNA1 is bound to the EBV origin. RPA is a heterotrimer, and the main contact with EBNA1 is formed through the 70 kDa subunit of RPA, the subunit which binds to ssDNA. We propose that this interaction between RPA and EBNA1 is an early step in activation of the latent origin of EBV.     

Time-Lapse Cinemicrographic Studies of X-Irradiated HeLa S3 Cells: I. Cell Progression and Cell Disintegration

Time-lapse cinemicrographs of synchronous HeLa S3 cells irradiated with 220 kv X-rays at various stages of interphase provided data for constructing pedigrees, measuring the duration of both generation cycles and mitoses, and scoring events associated with cell disintegration for up to seven postirradiation generations. The onset of the first mitosis after doses of 500 rads was delayed as expected from previous studies of the age dependence of “mitotic delay.” The interval between this first mitosis and the next was indistinguishable from that for unirradiated control cells, while the subsequent two generations were again prolonged, on the average, though not so severely as was the irradiated generation. The duration of mitosis was increased proportionally more than interphase. Cell disintegration took place by way of two morphologically distinct processes. In three-quarters of the cases the cells were rounded and apparently trapped in metaphase when they disintegrated; the remaining disintegrations occurred in spread, interphase cells. In cells disintegrating from the rounded configuration, the generation preceding disintegration was prolonged relative to that in cells which divided; in cells disintegrating from either configuration, the penultimate generation was also prolonged. The mitotic times were disproportionately increased in both of these generations. It is suggested that in this system X-ray damage is preferentially expressed as derangement of the mitotic process; such damage ultimately brings about permanent mitotic arrest in the majority of cells.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication

Human single-stranded DNA binding protein (human SSB) is a multisubunit protein containing polypeptides of 70, 34, and 11 kDa that is required for SV40 DNA replication in vitro. In this report we identify the functions of the SSB and its individual subunits in SV40 DNA replication. The 70 kDa subunit was found to bind to single-stranded DNA, whereas the other subunits did not. Four monoclonal antibodies against human SSB were isolated which inhibited SV40 DNA replication in vitro. The antibodies have been designated alpha SSB70A, alpha SSB70B, alpha SSB70C, and alpha SSB34A to indicate which subunits are recognized. Immunolocalization experiments indicated that human SSB is a nuclear protein. Human SSB is required for the SV40 large tumor antigen-catalyzed unwinding of SV40 DNA and stimulates DNA polymerases (pol) alpha and delta. The DNA unwinding reaction and stimulation of pol delta were blocked by alpha SSB70C, whereas the stimulation of pol alpha by human SSB was unaffected by this antibody. Conversely, alpha SSB70A, -70B, and -34A inhibited the stimulation of pol alpha, but they had no effect on DNA unwinding and pol delta stimulation. None of the antibodies inhibited the binding of SSB to single-stranded DNA. These results suggest that DNA unwinding and stimulation of pol alpha and pol delta are required functions of human SSB in SV40 DNA replication. The human SSB 70-kDa subunit appears to be required for DNA unwinding and pol delta stimulation, whereas both the 70- and 34-kDa subunits may be involved in the stimulation of pol alpha.     

Complete enzymatic synthesis of DNA containing the SV40 origin of replication

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.