Research Article: Changes in the Proteasome Pool during Malignant Transformation of Mouse Liver Cells

Date Published: April , 2010

Publisher: A.I. Gordeyev

Author(s): T.M. Astakhova, G.V. Delone, Yu.V. Lyupina, E.B. Abramova, I.V. Uryvaeva, N.P. Sharova.



Multiple forms of proteasomes regulate cellular processes by destroying proteins or forming
the peptides involved in those processes. Various pathologies, including carcinogenesis, are
related to changes in the functioning of the proteasome forms. In this study, we looked at the
changes in the pool of liver proteasomes during nodular regenerative hyperplasia and formation
of adenoma and hepatocellular carcinoma in mice treated with Dipin, followed by partial liver
resection. The relative content of various proteasome forms was determined using Western blot
analysis. The chymotrypsin–like activity of proteasomes was assessed from the hydrolysis
of the commercial Suc–LLVY–AMC substrate. It was found that
changes in the proteasome pool appeared already during the formation of diffuse nodules, the
changes being the increased expression of the X(β5) constitutive subunit and the
LMP7(β5i) and LMP2(β1i) immune subunits, accompanied by the increase of the total
proteasome pool and the decrease in the chymotrypsin–like activity. These changes were
more pronounced in hepatocellular carcinoma. The content of the total proteasome pool and the
LMP2(β1i) immune subunit and the chymotrypsin–like activity in adenoma were
intermediate compared to those in the samples of liver with diffuse nodules and carcinoma. In
addition, the level of the Rpt6 subunit present in the 19S proteasome activator was increased
in carcinoma. Our results indicate that nodular regenerative hyperplasia and adenomatosis may
be stages preceding carcinogenesis. We also conclude that there is a need to find signalling
pathways that change the expression of various proteasome subunits during carcinogenesis. The
19S proteasome activator, which is overexpressed in malignant tumours, can be a promising
target for the development of new anticancer drugs.

Partial Text

Understanding the molecular mechanisms underlying the malignant transformation of cells is of
ever vital importance. The new protein hydrolysis system discovered in the 1980s involving
proteasomes and affecting all cellular processes provided a new impulse to the studies of the
mechanisms of mammalian cell malignant transformation. Proteasomes, multisubunit
multiproteinase protein complexes, are present in mammalian organs and tissues in a multitude
of forms of different structures and physiological functions [1–4]. Proteasomes can be divided
into two groups—constitutive proteasomes and immunoproteasomes—depending on the
nature of their active protease subunits. The constitutive proteasomes contain two of each of
the X(β5), Y(β1) and Z(β2) subunits, possessing chymotrypsin–like,
caspase–like, and trypsin–like activity, respectively. The immunoproteasomes
contain the LMP7(β5i), LMP2(β1i), and LMP10(β2i) immune subunits instead of the
above–mentioned protease active subunits of the constitutive proteasomes. When foreign
proteins are hydrolysed by immunoproteasomes, the amount of antigen epitopes formed is several
times higher. The antigen epitopes are capable of incorporating into the Bjorkman gap of the
major histocompatibility complex class I molecules for further presentation to T lymphocytes.
In addition, immunoproteasomes participate in the regulation of the differentiation and
proliferation of some cell populations, perhaps, by producing biologically active peptides
[5, 6]. They are
also an essential part of the signalling pathway responsible for the quenching of oxidative
stress [7].

Reagents. The following reagents were used:
Suc–LLVY–AMC and MG132 (Sigma, USA),
anti–β–actin mouse mAb (Santa Cruz, Germany), anti–Rpt6
and anti–α1,2,3,5,6,7 mouse mAb, anti–X(β5),
anti–LMP7(β5i) and anti–LMP2(β1i) rabbit pAb (Biomol,
UK), anti–nNOS rabbit pAb (Abcam, UK), and ECL kit,
Hybond–ECL nitrocellulose membranes and peroxidase conjugated antibodies to mouse and
rabbit IgG (Amersham Biosciences, UK).

Histological study of mouse liver. The results of mouse liver histological
study performed 12 months after Dipin injection and partial liver resection are presented in
Fig. 1. Multiple nodules (benign tumours, microadenomas)
were revealed in the liver tissue (Fig. 1 a); they formed
during the diffuse nodular regenerative hyperplasia of hepatocytes. In addition, we detected
large benign tumours, adenomas, (Fig. 1 b) and malignant
tumours whose biological properties corresponded to hepatocellular carcinoma of the trabecular
type (Fig. 1 c) [21]. We performed a comparative study of the chymotrypsin–like activity
of the total proteasome pool and the content of various proteasome subunits in the tumour
samples and liver fragments with diffuse nodular hyperplasia versus liver samples of the
control animals.

The formation of hepatic nodular regenerative hyperplasia, adenomatosis, and carcinoma is
accompanied by changes in the proteasome pool, the changes having similarities, as well as
differences. The similarities are the increase in the content of immunoproteasomes and in the
total proteasome pool, and the decrease in the proteasome chymotrypsin–like activity in
all tumour types compared to the control samples. The difference is in the behavior of the 19S
proteasome activator content, which is increased only in hepatocellular carcimona.