Thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Science (Biochemistry), University of the Witwatersrand, Johannesburg, 1992An investigation has been made into aspects of the expression of
oncogenes in normally dividing cells and in hepatoceilular carcinoma (Hee).
HOC occurs commonly in Southern Africs, and thf1aetiology ·ofthis tumour
lsaseccieted with hepatitis a virus (HBV) infection.
c·erbA, c..mva and e-tos but not c~Ha..res mANA were elevatad in tumours
and adjacent hepatic tissue from the same petiEJ;htswhen compared to
normal liver. Amounts of Fos and MYQ prot~in in the liver tumour
specimens were else raised. The"e was some correlation between the
patients' serum a..fetoproteirt concentretlons, histological features of
tumour differentiatic)t"l, c..mvc and c40s r.ixpression.
expression of e-tas and c..myc has been reportec to be elevated after
stimulation of cells to alvlde, ,'1 occurs during liver r19ganeration. This was
corroborated by the findin~ that c-mvc, c·fo~· and c-jun mRNA
concentratlona "Jere increased it"! cultured 3T6 mouse fibroblasts following
treatment with alkaline medium aa a mitogenlo stimulus. The time course
of the expression of these oncogenes was similar to that reported after
gro\l'l/th factor sttmulation,
The H[~V X..gene ma\' be responsible for increased oncogene expression it'
YCC as a result of its documented trans activating properties. This vi!'a~
gene is unusual in that it has a codon preferanc";which is similar to that of
eukarvotic ceU genes. Also HBV may ha'V& evolved from ti similar ancestral
virus to that giving rise to retroviruses. These ideas suggest that the HBV
X·gene is a viral oncogene derived from a host homologue.
Low stringency Northern brot hybridisation using a X-gene probe
denlonstrated a murine transcrlpt in heart and thymus. Attempts to isolate
the sequence from mouse heart and thymus eDNA libraries ware
unsuccessful despite ext,~njve screening with sensitive probes (SP6
palymerfjsa and peR fab(':.lUed X~gen~~fragments). Conserved X~gene
\ . I sequences were also used fot the desigr:Jof primers in .~.peR bas£'d method
" . II
aimed at isolating a mammalian sequence. No sinnificant sequsnce
\\
homology was found bet\lveen the HBVI\X..gene and Ol\A ampllfle'd from
\1
l!
gen(llmic and eDNA I1br'srytemplate sou~\pes.The peR preducts ttppeared
to have been artef.,ots of arnplWaation. ~~n'IJreto detect the hQrtll.)logous
gene may have resu~ted from poo' complS,JIlentarity between the VIral ant!
\\
mammalian secuencec, 1\
\\
Non..~pecific amplification is commonly enct~unter&d when u1110 PCli'. A
qtJick asvmmatrlc re·ampW~catj(ii1 method I,?ssed on eXUOSilin of an
"
interm.uly' hybrfdising X·gelllapfimar we! davisQ\j to confirm FICRprOdu(,ts.
The l"n1ithodwas specific irlthat "ver~ single bas~ mlsmatohe betwsen the
internal primer and tem1>late re;.,ultad in fatJut~ of dete(;tabla \tUim$f
extension.GR 201
