D.S.R. Sarma

A growth-constrained environment drives tumor progression in vivo

We recently have shown that selective growth of transplanted normal hepatocytes can be achieved in a setting of cell cycle block of endogenous parenchymal cells. Thus, massive proliferation of donor-derived normal hepatocytes was observed in the liver of rats previously given retrorsine (RS), a naturally occurring alkaloid that blocks proliferation of resident liver cells. In the present study, the fate of nodular hepatocytes transplanted into RS-treated or normal syngeneic recipients was followed. The dipeptidyl peptidase type IV-deficient (DPPIV−) rat model for hepatocyte transplantation was used to distinguish donor-derived cells from recipient cells. Hepatocyte nodules were chemically induced in Fischer 344, DPPIV+ rats; livers were then perfused and larger (>5 mm) nodules were separated from surrounding tissue. Cells isolated from either tissue were then injected into normal or RS-treated DPPIV− recipients. One month after transplantation, grossly visible nodules (2–3 mm) were seen in RS-treated recipients transplanted with nodular cells. They grew rapidly, occupying 80–90% of the host liver at 2 months, and progressed to hepatocellular carcinoma within 4 months. By contrast, no liver nodules developed within 6 months when nodular hepatocytes were injected into the liver of recipients not exposed to RS, although small clusters of donor-derived cells were present in these animals. Taken together, these results directly point to a fundamental role played by the host environment in modulating the growth and the progression rate of altered cells during carcinogenesis. In particular, they indicate that conditions associated with growth constraint of the host tissue can drive tumor progression in vivo.

Dietary orotic acid, a new selective growth stimulus for carcinogen altered hepatocytes in rat

It was observed that orotic acid (OA), a precursor for pyrimidine nucleotide biosynthesis, when supplied exogenously at 1% level in the diet selectively stimulated the growth of hepatocytes modified by 1,2-dimethylhydrazine (1,2-DMH) to form gamma-glutamyltransferase (gamma-GT) (EC 2.3.2.2) positive islands. Increasing the duration of OA diet from 5 to 10 weeks resulted in an increase in the number of foci from 6 to 14/cm2. Rats that received the carcinogen and basal diet, however, developed only 1-2 foci/cm2. This unique effect of OA can be further accentuated by supplying a liver cell proliferative stimulus, such as a single necrogenic dose of CCl4.

Studies on the kinetics of expression of cell cycle dependent proto-oncogenes during mitogen-induced liver cell proliferation

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.

Induction of hepatic nodules in the rat by aristolochic acid.

Aristolochic acid (AA), used as an anti-inflammatory agent in the past, is known to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis and urinary bladder. However, despite the induction of DNA adducts in the liver, no carcinogenic potential of AA has been reported in the latter organ. The present study was based on the rationale that the lack of carcinogenicity of AA to the liver could be because this chemical may not be necrogenic at the doses examined and liver cell proliferation has been established as an essential component for initiation of liver carcinogenesis in the rat. The results indicated that AA is non-necrogenic to the rat liver. However, a single non-necrogenic dose of AA (10 mg/kg b.w., i.p.) given 18 hours after 2/3 partial hepatectomy initiated liver cell carcinogenesis. The initiated cells are promotable with 1% dietary orotic acid, a liver tumor promoter, to form glutathione-S-transferase 7-7 positive hepatic foci and nodules.

Effect of PSC 833, a potent inhibitor of P-glycoprotein, on the growth of astrocytoma cells in vitro

Malignant astrocytomas have been found to express P-glycoprotein (Pgp, mdr1 gene product). It was hypothesized that in addition to conferring multidrug resistance, Pgp is intimately associated with the development of astrocytomas. Accordingly, we studied the effect of PSC 833 (PSC, Novartis), a potent inhibitor of Pgp, on the growth of Pgp-expressing astrocytoma cells. The results showed that in all the cell lines tested, PSC (10-60 microM) inhibited the growth as well as induced cell death. Cells exposed to PSC exhibited DNA ladder characteristic of apoptosis. PSC-induced cell death could be reversed by Z-VAD-fmk, a general caspase inhibitor, indicating that PSC-induced cell death was characteristic of caspase-mediated apoptosis. These results suggest a novel therapeutic strategy in the treatment of malignant astrocytomas by inhibitors of Pgp.

Initiation of Experimental Liver Carcinogenesis by Chemicals: Are the Carcinogen Altered Hepatocytes Stimulated to Grow into Foci by Different Selection Procedures Identical?

The multi-stage nature of the carcinogenic process has been demonstrated in many tissues including skin (1–3), liver (4, 5), mammary gland (6), urinary bladder (7) and vagina (8). However, the molecular mechanisms underlying this complex process are not yet understood clearly. The development of several experimental model systems for the sequential analysis of chemical carcinogenesis in rat liver has opened up new avenues for investigating this complex process. Our experimental approaches have two main objectives (i) to understand the mechanisms underlying the initiation phase of liver carcinogenesis and (ii) to characterize the initiated hepatocytes stimulated to grow into enzyme altered foci by some of the selection procedures. The experimental design was guided by the hypothesis shown in Figure 1.

Influence of Orotic Acid on Multistage Hepatocarcinogenesis in the Rat: Resistance of Hepatocytes from Nodules to the Mitoinhibitory Effects of Orotic Acid

Abstract This study was designed to determine whether hepatocytes from hepatic nodules are resistant to the mitoinhibitory effects of orotic acid. Hepatic nodules were initiated in Fischer 344 male rats with 1,2-dimethylhydrazine. 2HCI (100 mg/kg ip) given 16 hr after two thirds partial hepatectomy and promoted by feeding a diet containing 1% orotic acid. Eight to 9 months later, when persistent nodules had developed, the rats were taken off the orotic acid diet and maintained on a semisynthetic basal diet for 2 to 5 weeks. The effect of orotic acid on the DNA synthesis in the hepatocytes isolated from hepatic nodules and from the surrounding nonnodular liver and from the age- and sex-matched control rats was studied. The results indicated that a dose of orotic acid (120 μM) that almost completely inhibited the transforming growth factor-α-induced DNA synthesis in hepatocytes from nonnodular surrounding liver and from age- and sex-matched control liver could not inhibit the DNA synthesis in hepatocytes from hepatic nodules. These results are consistent with the postulate that orotic acid may promote liver carcinogenesis by a differential mitoinhibition of normal hepatocytes while permitting the initiated hepatocytes to respond to growth stimuli and form hepatic nodules. However, it needs to be determined whether differential mitoinhibition of normal hepatocytes is the mechanism by which orotic acid promotes liver carcinogenesis.

Stimulation of DNA synthesis by rat plasma following in vivo treatment with three liver mitogens

The present study was undertaken to determine the effect of two different types of liver cell proliferative stimuli, namely compensatory regeneration and direct hyperplasia on DNA synthesis of normal and preneoplastic isolated hepatocytes. Platelet-poor plasma (PPP) isolated from male Wistar rats treated with three different hepato-mitogens, lead nitrate (LN), cyproterone acetate (CPA) and ethylene dibromide (EDB), or subjected to surgical partial hepatectomy (PH), was tested for its ability to stimulate DNA synthesis in normal and preneoplastic hepatocytes in primary cultures. Induction of DNA synthesis was detected as early as 30 min after CPA, EDB and PH administration and persisted up to 5 days after the LN administration. In addition, hepatocytes isolated from preneoplastic liver nodules were also able to respond in culture to the DNA synthesis stimulus induced by these factors.

Rapid and transient induction of c-fos, c-myc and c-Ha-ras in rat liver following glycine administration

Administration of glycine (2.5 mmoles/100 g., i.p.) results in an increased expression of several cell cycle dependent genes such as c-fos, c-myc and c-Ha-ras in the rat liver. The increased expression could be noticed as early as 20-40 minutes and declined by 2 hours following glycine administration. The rapid rise and decline in the mRNA levels of c-fos, c-myc and c-Ha-ras in response to glycine is of significance because in response to a wide variety of growth stimuli, these proto-oncogenes exhibit a temporal sequence in their expression; for example, the expression of c-fos precedes that of c-myc, which in turn precedes the increased expression of c-Ha-ras. The experimental model using a simple amino acid such as glycine will be useful in exploring some of the mechanisms of regulation of expression of these proto-oncogenes.

An earlier proliferative response of hepatocytes in γ-glutamyl transferase positive foci to partial hepatectomy

One of the hallmarks of initiated hepatocytes is their resistance to several hepatotoxins. This property forms the basis for their selective growth under conditions which are inhibitory to the non-initiated hepatocytes. Selective growth of initiated hepatocytes also occurs, albeit at a low level, in initiated rat liver without exposure to any known promoting regimen and/or in the absence of any known selective pressure to which initiated hepatocytes can possibly be resistant. This latter phenotypic property of initiated hepatocytes was further characterized by comparing the kinetics of response of hepatocytes in gamma-glutamyl transferase positive foci and in the surrounding liver to 2/3 partial hepatectomy both in the presence and in the absence of a promoting regimen. Male Fischer 344 rats (130-150 g) were initiated with a single dose of diethylnitrosamine and 1 week later they were placed on either a semi-synthetic basal diet or a promoting diet containing 1% orotic acid. Partial hepatectomy was performed 15 weeks after initiation and animals from both groups were killed at 12, 16, 20, 24, 30, 36, 48, 72 or 96 h after operation. Each animal received a pulse of 3H-labelled thymidine 1 h prior to killing. Autoradiographic studies revealed that hepatocytes in gamma-glutamyl transferase positive foci in the livers of rats fed the basal diet were significantly labelled at 16 h post-partial hepatectomy while surrounding hepatocytes were still virtually quiescent (LI 12.7 +/- 4.7 versus 1.2 +/- 0.5%, respectively). Higher labelling index in foci compared to the surrounding liver was also seen at 20 h post-PH (36.9 +/- 2.6 versus 21.5 +/- 2.4). Similar earlier response of hepatocytes in gamma-glutamyl transferase positive foci was also seen in initiated rats exposed to dietary orotic acid. In addition, orotic acid treatment appears to have imposed a slight delay on the entry of hepatocytes in the surrounding liver into S phase and thereby enhancing the differential of growth response between these two populations.