von Gruenigen, Vivian E.; Jamison, James M.; Gilloteaux, Jacques; Lorimer, Heather E.; Summers, Marcia; Pollard, Robert R.; Gwin, Carley A.; Summers, Jack L. Anticancer Res. 23(4) July-August 2003. 3279-3288.
Background: The objective was to evaluate the cytotoxic effect and mechanism of action of vitamins C (VC) and K3 (VK3) on ovarian carcinoma. Materials and Methods: Cytotoxicity assays were performed on ovarian cancer cell lines with VC, VK3 or a VC/VK3 combination. FIC index was employed to evaluate synergism. Flow cytometry was accomplished at 90% cytotoxic doses. Light, transmission electron microscopy
and DNA isolation were performed. Results: Antitumor activity was exhibited by both VC, VK3 and VC/VK3. VC/VK3 demonstrated synergistic activity. VC/VK3 may induce a G1 block in the cell cycle. Combined vitamin treatment resulted in cells that maintain apparently intact nuclei while extruding pieces of organelle-free cytoplasm. Degradation of chromosomal DNA was observed. Conclusion: Cell death (autoschizis) displayed characteristics of both apoptosis and necrosis. The cytotoxic effects observed may enable vitamins C and K3 to play an adjuvant role in the treatment of ovarian cancer.
|Analysis of Native Forms of Mitochondrial DNA by 2D
Gel Electrophoresis. Heather E. Lorimer
in "Methods in Molecular Biology: Mitochondrial DNA: Methods and Protocols." 2002 The Humana Press Inc., Totowa, N.J.
Ian J. Holt, Heather E. Lorimer, and Howard T. Jacobs
Cell, 100: 515-524, March 3, 2000
Analysis of mammalian mtDNA by two-dimensional agarose gel electrophoresis revealed two classes of replication intermediates. One was resistant to single-stranded nuclease digestion and displayed the mobility properties of coupled leading- and lagging-strand replication products. intermediates of coupled, unidirectional mtDNA replication were found in mouse liver and human placenta and were the predominant species in cultured cells recovering from transient mtDNA replication. Replication intermediates sensitive to single-strand nuclease were most abundant in untreated cultured cells. These are presumed to derive from the orthadox, strand asynchronous mode of mtDNA replication. These findings indicate that two modes of mtDNA replication operate in mammlaian cells and that changes in mtDNA copy number involve an alteration in the mode of DNA replication.
A TEST OF THE TRANSCRIPTION MODEL FOR BIASED
INHERITANCE OF YEAST MITOCHONDRIAL-DNA
Lorimer, H.E., Brewer, B.J., Fangman, W.L.
MolecularandCellularBiology 15: (9) 4803-4809 September 1995
Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA)
replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae
mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA
replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model
are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]).
These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-]
cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication
advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test
whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the
nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold.
Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings
between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not
preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-]
mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41.
Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results,
and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA
FOR RECOMBINATION JUNCTIONS IN THE SEGREGATION OF
MITOCHONDRIAL-DNA IN YEAST
Lockshon, D., Zweifel, S.G., Freeman-Cook, L.L., Lorimer, H.E., Brewer B.J., Fangman, W.L.
Cell81: (6) 947-955 June 16, 1995
In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if
there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed
using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes
relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that
encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger
proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of
mtDNA into a small number of cytological structures. This change in mtDNA structure can account far the
increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that
the level of unresolved recombination junctions influences the number of heritable units of mtDNA.
POLYOMAVIRUS AND SIMIAN-VIRUS-40 LARGE T-ANTIGENS PRODUCE
DIFFERENT STRUCTURAL ALTERATIONS IN VIRAL ORIGIN DNA
Bhattacharyya, S., Lorimer, H.E., Prives, C.
Journal of Virology 69: (12) 7579-7585 December 1995
Murine polyomavirus (Py) and simian virus 40 (SV40) encode homologous large T antigens (T Ags) and also
have comparable sequence motifs in their core replication origins. While the ability of SV40 T Ag to produce
specific distortions,within the SV40 core replication origin (ori) in a nucleotide-dependent fashion has been
well documented, little is known about related effects of Py T Ag on Py ori DNA. Therefore, we have
examined viral origin DNA binding in the presence of nucleotide and the resulting structural changes induced
by Py and SV40 T Ags by DNase I footprinting and KMnO4 modification assays. The structural changes in
the Py ori induced by Py T Ag included sites within both the AIT and early side of the core origin region,
consistent with what has been shown for SV40. Interestingly, however, Py T Ag also produced sites of
distortion within the center of the origin palindrome and at several sites within both the early and late regions
that flank the core ori. Thus, Py T Ag produces a more extensive and substantially different pattern of
KMnO4, modification sites than does SV40 T Ag. We also observed that both T Ags incompletely protected
and distorted the reciprocal ori region. Therefore, significant differences in the interactions of Py and SV40 T
Ags with ori DNA may account for the failure of each T Ag to support replication of the reciprocal ori DNA in
permissive cell extracts.
AND ACTIVE ATP HYDROLYSIS ARE NOT REQUIRED FOR
SV40 T-ANTIGEN HEXAMER FORMATION
Reynisdottir, I, Lorimer, H.E., Friedman, P.N., Wang, E.H., Prives, C.
JournalofBiological Chemistry 268: (33) 24647-24654 November 25 1993
ATP induces structural alterations in SV40 large T antigen and promotes changes in its interaction with the
viral replication origin. We have analyzed nucleotide-induced changes in T antigen structure in the absence of
origin DNA. Most preparations of immunopurified T antigen contain several discrete species ranging in size
from monomers through oligomers larger than hexamers. The predominant species consist of monomers and
dimers. Incubation of T antigen with ATP or dATP leads to a dramatic and rapid increase in the appearance of
T antigen hexamers. Weakly and nonhydrolyzable analogs of ATP are effective as well, indicating that
hexamer formation does not require active ATP hydrolysis. After incubation of T antigen with
[gamma-S-35]ATP, stable association of the labeled nucleotide with all detectable forms occurs. Removal of
greater than 80% of the T antigen phosphate residues does not significantly affect the formation of T antigen
hexamers, although changes in the distribution and mobility of the other species of T antigen are apparent.
Furthermore, T antigen synthesized in and purified from Escherichia coli and, therefore, presumably un- or
underphosphorylated, is capable of forming hexamers. Nucleotide-induced T antigen hexamer formation thus
appears to require neither protein phosphorylation nor active ATP hydrolysis.
UNUSUAL PROPERTIES OF A REPLICATION-DEFECTIVE MUTANT SV40 LARGE
Lorimer, H.E., Reynisdottir, I., Ness, S., Prives, C.
Virology192: (2) 402-414 FEB 1993
THE DNA-BINDING PROPERTIES OF POLYOMAVIRUS LARGE T-ANTIGEN ARE
ALTERED BY ATP AND OTHER NUCLEOTIDES
Lorimer, H.E., Wang, E.H., Prives, C.
JournalofVirology 65: (2) 687-699 Feb. 1991
We have examined the influence of ATP on the DNA-binding properties of polyomavirus large T antigen (Py
TAg). Utilizing nitrocellulose filter binding, DNase I footprinting, and gel mobility shift assays, we observed
that ATP increased Py TAg binding to DNA fragments containing either all Py TAg-binding sites (whole
origin) or those sites within (core origin) or adjacent to (early) the origin of replication. Even nonspecific
binding to DNA fragments lacking Py TAg-binding sites was increases somewhat by ATP. Binding to the core
origin was increased to a greater extent than binding to other DNA fragments tested. Gel band mobility shift
assays revealed that ATP increased the production of core origin-specific Py TAg-DNA complexes of high
molecular weight. ATP stimulation depended on the presence of MgCl(2). Other nucleotides and
nonhydrolyzable ATP analogs also increased Py TAg binding to the core origin but to various degrees: ATP,
dATP, 5'-adenylyl imidodiphosphate (AMPPNP) > 5'-adenylyl methylenediphosphate (AMPPCP) > dCTP >
UPT > TTP. GTP and dGTP did not increase DNA binding by Py TAg. The rates of association and
disassociation of Py TAg with all the DNA fragments were altered by the presence of ATP. DNase I
footprinting showed that ATP extensively extended the region protected within the core origin and also
produced a distinctive DNase I-hypersensitive site on the late strand at nucleotides 5255 to 5262
of Polyomavirus Large T Antigen by Using a Baculovirus Vector.
Rice WC.; Lorimer HE.; Prives C.; Miller LK.
Journal of Virology 61: (5) 1712-6 May 1987
A gene encoding the large T antigen of polyomavirus was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus so that gene expression was under the control of the strong, very late polyhedrin gene promoter. Significantly more large T antigen was produced in recombinant virus-infected insect cells than was observed in polyomavirus-transformed mouse cells. The insect-derived T antigen exhibited polyomavirus origin-specific DNA binding. The baculovirus expression system provides a convenient source of T antigen for in vitro studies.