Zhi-Hua Ran
Dr. med.
Investigation of molecular mechanisms underlying the oncosuppressive activity of
Parvoviruses H-1 and MVM
Date of birth: 12.07.1962
A levels: 07.1980
Studies: Medicine. 09.1980- 07.1985
Clinical studies: 09.1983- 07.1985
Practical year: 09.1984- 07.1985
Graduation: 07.1985. Shanghai Second Medical University
Subject: DKFZ-ATV
Tuitor: Prof. Jean Rommelaere, Ph.D
In order to better understand parvoviral oncolytic effects, the molecular mechanisms
underline virus-cells interactions have been investigated through three different approaches
both in vivo and in vitro.
1. To account for the oncolytic activity of parvoviruses, a possible link was sought
between, on the one hand, expression of the tumor-suppressing protein p53 and, on the other
hand, cytotoxicity associated with H-1 virus infection. To this end, a wild-type (wt) p53
inducible system derived from an endogenous p53- hepatoma cell line was chosen for the
present study. Wt p53 activity can be induced in these cells (designated 4P) upon addition the
inducer, 4-OH tamoxifen. At the optimal concentration of 4-OH tamoxifen (750 nM), the
induction of p53 expression was found to have no detectable effects on the growth properties
of uninfected cells. This allowed the inducible system to be used to analyze the effects of p53
on cell killing by parvovirus H-1. Our data indicate that in this system, p53 expression was
not associated with a significant change in the sensitivity of cells to the cytotoxic action of
parvovirus H-1. Therefore, no definite conclusion can be drown concerning a possible
influence of p53 on cell susceptibility to parvovirus infection. Indeed the cellular system
tested can be distinguished by its unaffected growth in the presence of p53 and by its peculiar
resistance to some DNA-damaging agents, and may not be representative of the normal
functioning of p53.
2. The question as to the tissue-specificity and developmental regulation of the MVM P4
promoter was addressed by means of a transgenic approach. This is an important issue for the
understanding of oncolysis, because promoter P4 directs the transcription unit encoding the
parvoviral cytotoxic proteins (designated NS). Three transgenic mouse lines were studied, two
in which the LacZ reporter gene was placed under the direct control of P4 (P4-LacZ
construct), and one harbouring the (P4-NS-P38-LacZ) construct in which LacZ expression is
driven by the NS-inducible parvoviral promoter P38. The P4-LacZ transgenic mouse lines
were tested for P4 activity in vivo. In both cases, the P4 promoter was found to remain silent
during embryogenesis. In order to confirm and further investigate the possible reasons for
such a lack of transgene expression, 27 primary cell cultures were prepared from embryos
derived from all three transgenic mouse lines. Normal 3T3 cell lines and their SV40
transformed derivatives were further established from these primary cell cultures. The various
cell lines allowed P4 promoter activity to be measured in vitro both qualitatively (Bluo-gal
staining) and quantitatively (chemiluminescent reporter assays). Again, the P4 promoter was
found to be silent in both normal and SV40-transformed 3T3 cells. Two lines of evidence
suggested that the transgenes were present. Firstly, a positive signal was obtained by DNA
hybridization; secondly, NS1 proteins produced after infection of P4-NS-p38-LacZ transgenic
cell cultures with MVM led to activation of the P38 promoter and expression of the reporter
gene. Other observations suggest that our failture to detect P4 activation during
embryogenesis is likely to be due to the strain of mice studied and/or the pattern of transgene
integration in the animals tested. Our work shows that the proposed approach is feasible and
should be extended to other mice.
3. New insights into the mechanisms by which parvovirus H-1 causes cell death was
obtained in Hela cells and in SV40-transformed rat fibroblasts (P1) that were made inducible
for the survival factors bcl-2 or hsp27. Through morphological analysis (Hoechst / PI dual
staining), necrotic cell death was observed in P1 cells after H-1 virus infection. H-1 virus-
induced cell killing occured via a bcl-2 and hsp27 insensitive pathway in the P1 cell system.
Biochemical (PARP cleavage) and morphological (Hoechst / PI dual staining) analyses
indicated that necrotic death also occured in Hela cells after H-1 virus infection, although
some changes characteristic of apoptosis may be triggered at early times. H-1 virus infection
of Hela cells was accompanied by rapid depletion of intracellular NAD stores. Stimulation of
NAD production had no detectable effect on cell death. In contract, inhibition of NAD-
consuming enzymes were found to shift the mode of death from necrosis to apoptosis in a
fraction of Hela cells. These data show that parvoviruses can kill cells by both necrotic and
apoptotic processes, and point to ADP ribosylation as a new parameter for the decision on
whether cells will get involved in one or the other death pathway.
Bachelor’s degree: 09.1980-07.1985. Shanghai Second Medical University
Master’s degree: 09.1990-07.1994. Snanghai Second Medical University
