Improved adenovirus vector provides herpes simplex virus ribonucleotide reductase R1 and R2 subunits very efficiently.

We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the activity of the ectopic major late promoter (MLP) in recombinant Ad. In 293 cells infected with helper-free Ad recombinants generated with the pAdBM5 transfer vector, both herpes simplex virus (HSV) ribonucleotide reductase R1 and R2 subunits represent the most abundant polypeptides, accounting for as much as 15-20% of total cellular proteins. Our data suggest that this level of expression is probably very close to the upper limit of the system. Furthermore, when compared to the widely utilized baculovirus (Bac)/Sf9 expression system, the improved Ad vector showed a better performance for the production and purification of active HSV-2 ribonucleotide reductase R1 and R2 subunits. The R2 subunit was about 5-fold more abundant in recombinant Ad-infected 293 cells than in Bac-infected Sf9 cells while the R1 subunit was produced at roughly similar levels with either system. However, the amount of active soluble R1 obtained from recombinant Ad-infected 293 cells was at least 5 times higher because most of the R1 produced in Sf9 cells was insoluble.

Abnormal delayed hypersensitivity response to antigens of Candida albicans in alloxan-diabetic mice.

Three antigens of Candida albicans were comparatively evaluated to their ability to elicit delayed hypersensitivity (DH) responses in the mouse footpad test, using alloxan-diabetic and normal mice which were primed with heat-killed C. albicans in complete Freund adjuvant. These antigens were: (1) a preparation of sonically disrupted heat-killed cells; (2) a preparation of soluble cytoplasmic material remaining in the supernatant of a broken-cell suspension centrifuged at 100,000 g; (3) a preparation obtained by extraction of pulverized defatted cells with dilute phenol and sodium bicarbonate in water. After separation by polyacrylamide gel electrophoresis, the major components of soluble cytoplasmic material and dilute phenol extract were identified as a 43-kD protein, and glycoproteins of 21, 27 and 38 kD, respectively. Fifty-eight CD-1 outbred mice, which had received a single intravenous injection of alloxan followed by a 28-day rest period, were randomized with normal littermates to distinct experimental groups. Seven days after sensitization, mice were injected with one of the antigens in the right rear footpad and saline in the left rear footpad and the net specific increase in footpad thickness determined 24 and 48 h later. All three antigens elicited significant responses in sensitized normal mice. The responses of sensitized diabetic mice were clearly inferior to those of sensitized normal mice when heat-killed cells and soluble cytoplasmic material were used. Dilute phenol extract elicited equivalent responses at 24 and 48 h in both primed diabetic and normal mice.

Reduction of c-myc expression correlated with E1a expression but not with the transformed phenotype.

The adenovirus E1a oncogene has both positive and negative regulatory effects on the expression of a variety of host genes. Both type of effects have been reported for certain cell cycle genes such as c-myc. To study the potential role of c-myc in adenovirus transformation, we have assessed the steady-state levels of c-myc mRNA after serum stimulation in genetically related transformed or non-transformed cell lines in the presence or absence of E1a. Serum stimulated the accumulation of stable c-myc mRNA only in cell lines which did not express E1a. Therefore under the present assay conditions, E1a had a negative effect on the steady-state level of c-myc mRNA. Surprisingly, this effect was independent of the transformed phenotype.

Drug-induced revertants of adenovirus-transformed cells: retransformation by 5-azacytidine without reactivation of E1a.

We have isolated drug-resistant variants from adenovirus-transformed rat cells that had concomitantly lost their transformed phenotype. Our aim was to determine the reason for reversion, to attempt retransformation with 5-azacytidine (5-AzaC) and to study the mechanism of retransformation. Of the three cell lines studied, one (G4F) had lost the integrated E1a genes, whereas the other two (G2a and G5) failed to synthesize E1a RNA or proteins. Incubation of these cell lines with 3 microM-5-AzaC for 2 days, followed by passaging in the absence of drug, gave rise to transformed foci in all of the cell lines. The efficiency of transformation was typical of each cell line. Surprisingly, retransformation was not accompanied by the reappearance of detectable levels of E1a gene activity in the G2aAza and G5Aza cell lines. In search of a mechanistic explanation for the loss of gene activity in the revertants and its reappearance in the retransformants, we examined the state of methylation of the E1a gene region in these cells. Neither the E1a promoter nor its upstream region was methylated in the revertants or the 5-AzaC retransformants. These results suggest that E1a transcription was suppressed by mechanisms other than DNA methylation and that 5-AzaC could retransform these cells without lifting the E1a-suppressed state.

Isolation and uptake characteristics of adenovirus transformed cell revertants resistant to the antiproliferative effects of methylglyoxal bis(guanylhydrazone).

Four independent variant cell lines resistant to the toxic action of methylglyoxal bis(guanylhydrazone), an anticancer drug and inhibitor of polyamine synthesis, have been isolated by single step selection from an adenovirus type 2-transformed rat brain cell line. Drug-resistance was accompanied by loss of tumorigenic potential in athymic nude mice. MGBG resistance was attributable to decreased drug uptake.