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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 9, 2000 as doi:10.1096/fj.00-0445fje. |
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Division of Gastroenterology and Liver Diseases, USC Liver Disease Research Center, USC School of Medicine, Los Angeles, California 90033, USA
2Correspondence: Division of Gastrointestinal and Liver Diseases, HMR Bldg., 415, Department of Medicine, USC School of Medicine, 2011 Zonal Ave., Los Angeles, CA, 90033, USA. E-mail: shellylu{at}hsc.usc.edu
SPECIFIC AIMS
GSH level increases in hepatocytes during active proliferation. Whether this occurs in human hepatocellular carcinoma (HCC) is unknown. The significance of the increase in GSH during hepatocyte proliferation is also unknown. The aims of the study were twofold: 1) to examine whether the GSH level is increased in HCC and determine the mechanism, and 2) to examine the significance of the increase in GSH during the growth of both normal and cancerous liver.
PRINCIPAL FINDINGS
1. GSH levels and expression of GSH synthetic enzymes are increased
in HCC
GSH levels (in nmol/mg protein) doubled in HCC as compared to
normal liver (normal liver = 22.4±3.5, HCC = 41.9±7.2;
results represent mean±SE from n=6 each,
P<0.02 by unpaired t test). The steady-state GCS
heavy subunit (GCS-HS) mRNA level is increased (298±29% of normal
liver by densitometric analysis, P<0.05). In contrast, the
steady-state GCS-light subunit (GCS-LS) mRNA level is unchanged. The
protein level of GCS-HS also increased comparably as the mRNA level
(241±31% of normal liver by densitometric analysis,
P<0.05). The steady-state GSH synthetase (GS) mRNA level is
also increased in HCC (254±32% of normal liver by densitometric
analysis). Nuclear run-on assay showed that the molecular mechanism for
the increase in GCS-HS and GS mRNA levels is largely increased
transcription (GCS-HS = 215±13% of normal, GS = 190±14%
of normal liver by densitometric analysis, P<0.05 by
unpaired t test) (Fig. 1
).
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2. There is increased nuclear binding activity to antioxidant
response element 4 (ARE-4), nuclear factor kappa B (NF-
B), and
activator protein 1 (AP-1) elements of the GCS-HS promoter in HCC
To investigate possible molecular mechanisms of increased GCS-HS
transcription, we examined nuclear binding activities of three
important cis-acting elements, ARE-4, NF-B, and AP-1,
identified in the promoter of GCS-HS. There is increased nuclear
binding to ARE-4, NF-B, and AP-1 elements of the GCS-HS promoter in
HCC.
3. Increased GSH level facilitates growth of HepG2 cells
To examine the role of GSH in the growth of liver cancer cells, we
cultured HepG2 cells in sulfur amino acid-deficient medium supplemented
with 10% fetal bovine serum, 1 mM methionine, and varying
concentrations of cystine (0 to 0.2 mM). GSH level was also increased
by treating cells with GSH ethyl ester (GEE, 1 mM), which bypasses the
GSH synthetic pathway. In some experiments, the GSH level was lowered
by adding buthionine sulfoximine (BSO, 1 mM) to the medium for 24 h. These treatments did not result in increased cell lysis as measured
by lactate dehydrogenase release into culture medium.
Cell growth increased with increasing cystine concentration. Addition of GEE (1 mM) also resulted in increased growth. On the other hand, addition of BSO (1 mM) resulted in marked inhibition in growth. Increasing concentrations of cystine in culture medium resulted in increasing GSH levels and rates of growth. As expected, GEE treatment increased and BSO treatment decreased GSH levels and rates of growth. There is a strong linear correlation between GSH levels and rates of growth (R=0.9, linear regression analysis).
To see whether decreased GSH in HepG2 cells affect DNA synthesis, cells were treated with BSO for 24 h and DNA synthesis was measured by quantitating the incorporation of 3H-thymidine into DNA during the last 6 h of treatment. As expected, DNA synthesis in BSO-treated cells was significantly lower than controls (control = 3708±177 dpm/µg DNA, BSO = 2403±89 dpm/µg DNA; results represent mean±SE from three separate determinations, P<0.05).
4. Both a normal baseline hepatic GSH level and a subsequent
increase in GSH are required for normal liver regeneration to occur
We showed previously that GSH and cysteine levels doubled 12 h after 2/3 partial hepatectomy (PH) and prior to the onset of DNA
synthesis. Here we examined the effect of preventing the increase in
liver GSH level on subsequent liver regeneration as well as the effect
of depleting liver GSH on liver regeneration. GSH was depleted and
synthesis inhibited by BSO. Two different regimens were used. In the
first regimen (BSO1), animals received BSO 900 mg/kg i.p. 45 min prior
to PH, repeated 12 h later. This was aimed at keeping the starting
liver GSH level relatively normal, but the increase in GSH after PH was
prevented. In the second regimen (BSO2), animals received BSO 667 mg/kg
i.p. 6 h prior to PH, repeated twice every 12 h. This
resulted in a 50% lower starting liver GSH level and prevented the
increase in GSH after PH.
If the starting liver GSH (in µmol/g) was kept relatively normal (BSO1 = 4.97±0.26, 2/3 PH = 6.2±0.23, results represent mean±SE from five to nine animals for each condition, P<0.05) and the increase in GSH (in µmol/g) at 24 h post-PH was prevented (BSO1 = 2.63±0.5, 2/3 PH = 8.37±0.31, results represent mean±SE from five to nine animals for each condition, P<0.05), there was a 33% inhibition in DNA synthesis (in dpm3H-thymidine incorporation/µg DNA) (BSO1 = 23.7±4.72, 2/3 PH control = 35.5±4.68, sham controls = 6.35±0.58, results represent mean±SE from five to nine animals for each condition, P<0.05). However, if the starting liver GSH (in µmol/g) was already 50% depleted (BSO2 = 2.92±0.53) and the increase was also prevented (BSO2 = 1.95±0.33), then DNA synthesis was inhibited by nearly 60% (BSO2 = 14.6±2.52). Finally, we examined what effect the lower hepatic GSH level and prevention of the increase in GSH had on the course of liver regeneration. Rats were treated with BSO2 or vehicle and followed for up to 7 days. Although the initial hepatic GSH levels were much lower in the BSO-treated group, by the third day after PH the levels were not significantly different from the control group. There was also no toxicity of BSO, since both groups of rats lost and gained comparable amounts of weight by 3 and 7 days after PH. Liver regeneration was complete by the seventh day in the control group but 20% lower in the BSO-treated group. Similarly, total liver DNA amount was 20–28% lower in the BSO-treated group at 3 and 7 days after PH.
CONCLUSIONS AND SIGNIFICANCE
GSH serves numerous important functions including antioxidant defense, storage of cysteine, maintenance of intracellular redox state, and modulation of cell growth. Many studies involving lymphocytes and fibroblasts showed that an increased GSH level was associated with an early proliferative response and was essential for the cell to enter the S phase. We previously showed that GSH level is increased when rat hepatocytes enter periods of rapid growth, such as plating under low cell density in primary culture or after 2/3 PH. However, the significance of the increase in GSH was not examined.
GSH has been found to be elevated in a number of drug-resistant tumor cell lines and in tumor cells isolated from patients whose tumors are clinically resistant to drug therapy. These tumors are of prostate, ovarian, lung, or colorectal origins. Increased GCS activity and GCS-HS gene transcription were found in the majority of these resistant tumor cells. Whether GCS-LS is also altered in tumor is controversial. Expression of GS has not been examined in any tumor. Whether the GSH level is increased in human liver cancer is unknown. This is a clinically important topic since there is currently no effective medical therapy against liver cancer.
In six consecutive HCC specimens, we found the steady-state GSH level doubled as compared to normal liver. Since our previous work found increased GSH biosynthesis accounted for the increase in GSH levels, we focused on the expression of the GSH synthetic enzymes. Similar to what we observed in the regenerating rat liver, the steady-state GCS-HS mRNA level was significantly increased whereas that of the GCS-LS was unchanged. We also found a comparable increase in the mRNA level of GS. Recently we have shown that GS is induced by many of the same agents that induce the GCS subunits. In general, agents that increased the expression of both subunits also increased the expression of GS; however, agents that increased the expression of GCS-HS alone had no influence on GS, with one notable exception, which is liver regeneration. Induction of GS appears to further enhance the capacity to synthesize GSH. In HCC, the increase in both GCS-HS and GS mRNA levels occurs predominantly at the transcriptional level, as demonstrated by the nuclear run-on assay.
We next examined possible molecular mechanisms for the transcriptional
up-regulation of GCS-HS in HCC. Previous studies showed that an AP-1
element located at -269 to -263 of GCS-HS is important in mediating
the effect of oxidative stress on GCS-HS expression. On the other hand,
Mulcahy et al. described a critical distal ARE element (ARE-4) that
mediated constitutive and ß-naphthoflavone inducible expression of
GCS-HS in HepG2 cells. More controversial is the role of a NF-B
consensus element located at -1099 to -1091 of GCS-HS. In human HCC,
there are increased nuclear binding activities to all three sites as
demonstrated by electrophoretic mobility shift assay using specific
oligonucleotide probes. However, whether or not they contribute to the
GCS-HS transcriptional activation remains to be determined.
The human GCS-LS promoter also contains ARE and AP-1 elements that were shown to be important for ß-naphthoflavone-induced increased GCS-LS expression in HepG2 cells. Despite increased binding activities to ARE and AP-1 elements in HCC, GCS-LS expression was not induced. We have also reported this dissociation after ethanol treatment, another condition where ARE and AP-1 nuclear binding activities increased but only GCS-HS was induced. Galloway and McLellan have also shown that the inducible expression of GCS-LS by t-butyl hydroquinone in HepG2 cells was not dependent on either ARE or AP-1. Thus, the roles of the ARE and AP-1 elements in the transcriptional regulation of GCS-LS remain unclear.
The role of GSH in the growth of liver cancer cells was next examined using HepG2 cells. GSH was varied by controlling the amount of cystine present in culture media. As expected, cell GSH increased as cystine concentration increased. This also resulted in increased growth rates. To see whether the effect on cell growth was due to a change in GSH, GSH was increased by treating cells with GEE or decreased by treating cells with BSO. Changes in cell growth and DNA synthesis paralleled changes in GSH levels, suggesting a causal relationship between the two.
Finally, we examined whether an increase in GSH is important for normal
liver regeneration to occur. We examined the effect of preventing the
increase in GSH as well as lowering the baseline GSH and preventing the
subsequent increase; many patients with chronic liver diseases and
diabetic mellitus have lower hepatic GSH levels, so whether a lower
hepatic GSH level hinders liver regeneration becomes an important
question. If the starting liver GSH level was kept relatively normal
and the increase in GSH prevented, there was a 33% inhibition in DNA
synthesis. However, if the starting liver GSH level was already 50%
depleted and the increase was also prevented, then DNA synthesis was
inhibited by nearly 60%. The inhibition on DNA synthesis at 24 h
is largely exerted on the regenerating hepatocytes since DNA synthesis
in the regenerating hepatic nonparenchymal cells lags behind by 
24 h. This is the first indication that normal GSH level and a
subsequent rise in GSH may both be important for normal liver
regeneration to occur.
The exact molecular mechanism of how a change in GSH resulted in a change in cell growth remains unclear. Possibilities include modulation of DNA synthesis by maintaining reduced glutaredoxin or thioredoxin, which are required for the activity of ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis. Alternatively, an increase in the cellular GSH content may change the thiol-redox status of the cell that is proportional to [GSH]2/[GSSG]. A change in the redox state may then affect the expression or activity of factors important for cell cycle progression. Further studies will be required to examine these possibilities.
In conclusion, we have shown for the first time that GSH level is
increased in human HCC as a result of increased expression of both
GCS-HS and GS at the transcriptional level. In the case of GCS-HS,
increased binding activity to three important cis-acting
elements occurs in HCC and may contribute to its transcriptional
up-regulation. Increased GSH level facilitates the growth of liver
cancer cells. Finally, a normal baseline hepatic GSH and a subsequent
increase may both be important for normal liver regeneration to occur.
Figure 2
summarizes the main
findings of this work.
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ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grants DK-45334 and Professional Staff Association Grant #6–268-0–0, USC School of Medicine. HepG2 cells, HCC, and normal liver specimens were provided by the Cell Culture Core of the USC Liver Disease Research Center (DK48522).
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0445fje To cite this
article, use (November 9, 2000) FASEB J. 10.1096/fj.00-0445fje 
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