p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells.

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.

A novel mechanism for chaperone-mediated telomerase regulation during prostate cancer progression.

Telomerase activity has been detected in >85% of all malignant human cancers, including 90% of prostate carcinomas. Using a well-characterized experimental prostate cancer system, we have found that telomerase activity is notably increased (>10-fold) during tumorigenic conversion. Expression profiles of the telomerase components (hTR and hTERT) revealed no substantive changes, which suggests a nontranscriptional mechanism for increased activity. Because the hsp90 chaperone complex functionally associates with telomerase, we investigated that relationship and found that along with telomerase activity, a number of hsp90-related chaperones are markedly elevated during transformation, as well as in advanced prostate carcinomas. Using the nontumorigenic cell protein extract as the source of telomerase, addition of purified chaperone components enhanced reconstitution of telomerase activity, which suggests a novel mechanism of increased telomerase assembly via a hsp90 chaperoning process during prostate cancer progression.

Stable association of hsp90 and p23, but Not hsp70, with active human telomerase.

The ribonucleoprotein telomerase holoenzyme is minimally composed of a catalytic subunit, hTERT, and its associated template RNA component, hTR. We have previously found two additional components of the telomerase holoenzyme, the chaperones p23 and heat shock protein (hsp) 90, both of which are required for efficient telomerase assembly in vitro and in vivo. Both hsp90 and p23 bind specifically to hTERT and influence its proper assembly with the template RNA, hTR. We report here that the hsp70 chaperone also associates with hTERT in the absence of hTR and dissociates when telomerase is folded into its active state, similar to what occurs with other chaperone targets. Our data also indicate that hsp90 and p23 remain associated with functional telomerase complexes, which differs from other hsp90-folded enzymes that require only a transient hsp90.p23 binding. Our data suggest that components of the hsp90 chaperone complex, while required for telomerase assembly, remain associated with active enzyme, which may ultimately provide critical insight into the biochemical properties of telomerase assembly.

Expression of c-kit (CD117) in benign and malignant human endometrial epithelium.

BACKGROUND: The proto-oncogene c-kit encodes a tyrosine kinase receptor (CD117) with a molecular weight of 145 kd. Previous studies, predominantly utilizing immunohistochemistry, have led to contradictory findings regarding the expression of CD117 in the endometrium. To help resolve this issue, we analyzed a series of benign and malignant endometrial tissues using both immunohistochemistry and Western blot analysis. OBJECTIVE: To examine the expression of CD117 in benign and malignant human endometrial tissues. METHODS: The expression of CD117 in 35 benign endometrial tissues (7 hyperplastic, 14 proliferative, 14 secretory) and 10 endometrioid carcinomas was investigated by immunohistochemistry (clone K45 monoclonal antibody). Immunoprecipitation (clone K69 monoclonal antibody) followed by Western blotting (clone K45 monoclonal antibody and clone 1.D9.3D6 monoclonal antibody) was performed to confirm CD117 expression. RESULTS: Fifty-seven percent of the hyperplasias, 93% of proliferative endometria, and 79% of secretory endometria immunostained positively for CD117. In benign endometria, epithelial staining tended to be more intense in the hyperplastic and proliferative endometria as compared to the secretory endometria, whereas endometrial stromal cells were not immunoreactive. Of the 10 frozen endometrial tissues analyzed by immunohistochemistry, 4 of 9 endometrioid carcinomas and a single case of an endometrioid polyp developing in association with a carcinoma expressed CD117. Immunoprecipitation followed by Western blot analysis confirmed expression of full-length CD117 in an endometrial polyp and carcinoma, and revealed a correlation between levels of immunoprecipitated CD117 and immunohistochemical staining intensity. CONCLUSIONS: Benign and malignant endometrial tissues express CD117. Our data suggest (a) a possible relationship between estrogen and CD117 expression in benign endometrium and (b) potential involvement of this growth factor receptor in endometrial carcinogenesis.

Telomerase and telomere stability: a new class of tumor suppressor?

Introduction of telomerase into normal cells provides telomere maintenance and an extended cellular life span, establishing the critical role of telomere attrition in cellular senescence. Additional data surrounding this observation suggest that expression of telomerase renders these "mortal" cells genomically stable with decreased frequencies of mutation, ultimately leading to continued proliferation without signs of changes typically associated with progression to a cancer-like phenotype. Interestingly, oncogenic insult after exogenous telomerase expression does not result in cellular transformation, yet addition of an oncogene first followed by telomerase does transform cells. Taken together, these results imply that order of addition is important for telomerase-mediated genomic protection and that telomerase expression is critical for the transformation process. The hypothesis proposed here is that telomerase, via its function in telomere stabilization, is capable of protecting cells from acquiring the required mutations and genomic instability necessary for malignant transformation, suggesting that telomerase is not an oncogene but may act as a novel class of tumor suppressor.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis.

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

"Intestinal-type" of adenocarcinoma preferentially induced in right/caudate liver lobes of rats treated with furan.

Short-term chronic exposures of rats to furan were recently found by us to preferentially induce a unique liver lobe pattern of development of small intestinal metaplasia and subsequent cholangiofibrosis, being essentially localized to the caudate and right liver lobes (L. W. Elmore, and A. E. Sirica, Cancer Res., 51: 5752-5759, 1991). We now demonstrate the preferential development of primary hepatic adenocarcinomas exhibiting small intestine mucosal cell differentiation, which have arisen at 70 to 90% incidences from the right/caudate liver lobes of Fischer 344 adult male rats by 16 months after their receiving furan by gavage at a daily dose of 30 mg/kg of body weight, five times a week, for 9, 12, and 13 weeks, respectively. In contrast, the incidences of primary hepatocellular carcinomas that developed in the furan-treated rats ranged from 0 to 20%, with the two hepatocellular carcinomas observed to be originating from the median/left liver lobes. Twenty-six of 27 hepatic adenocarcinomas analyzed exhibited glands containing on average 30.2% goblet cells, 2.1% Paneth cells, and 0.5% serotonin-positive neuroendocrine cells. Phenotypically, the glandular epithelial cells of the furan-induced intestinal-type adenocarcinomas were immunohistochemically positive for cytokeratin 19, but exhibited a heterogeneous pattern of immunohistochemical staining for gamma-glutamyl transpeptidase and showed no detectable immunostaining for transforming growth factor alpha. In addition, many of the glandular structures within these primary hepatic adenocarcinomas showed evidence of basement membrane disruption, as demonstrated by both electron microscopy and immunohistochemical staining for basement membrane laminin. While these intestinal-type adenocarcinomas appeared to have spread intrahepatically, none showed evidence of extrahepatic metastases. However, six of eight randomly selected adenocarcinomas grew progressively and retained their intestinal pattern of differentiation following serial transplantation into the fat pads of young adult Fischer 344 recipient rats. In this study, we also observed one primary hepatic cholangiocarcinoma that was characterized by a more native biliary rather than intestinal-type of differentiation. Interestingly, this was the only primary liver cancer observed by us to exhibit extrahepatic metastasis. In conclusion, our current findings clearly indicate that the small intestinal metaplasia and subsequent cholangiofibrosis developing early in the right/caudate liver lobes of furan-treated rats do not simply reflect reactive changes, but strongly correlate with the high incidences of intestinal-type of primary hepatic adenocarcinoma that occurs in the right/caudate liver lobes of rats after long-term exposures to furan.

Sequential appearance of intestinal mucosal cell types in the right and caudate liver lobes of furan-treated rats.

Furan rapidly induces in rat liver a unique, lobe-specific pattern of development of intestinal metaplasia and associated cholangiofibrosis. To establish early cell-precursor relationships in the genesis of this cholangiofibrosis, a time-course study was conducted in which young adult male Fisher 344 rats received furan by gavage at a daily dose of 45 mg/kg body wt over a 32-day treatment period. An analysis of individual liver lobes obtained at different time points from these animals during furan treatment revealed the following sequence of histopathological changes, which were located mainly in the right and, to a lesser extent, the caudate liver lobes: day 1, severe hepatonecrosis in zone 3 extending into zone 2 of the liver acini; days 3 to 5, presence of a diffuse inflammatory cell infiltrate in hepatonecrotic areas, which gradually resolved; day 7, prominent bile ductule hyperplasia in tissue sections exhibiting marked loss of normal liver parenchyma; day 9, continued replacement of injured liver by hyperplastic bile ductule tissue, which now contained occasional metaplastic glandular structures composed of columnar basophilic epithelial cells; days 12 to 16, increasing cell diversity in the developing metaplastic glands, with the sequential appearance of goblet cells, Paneth cells and serotonin-positive neuroendocrine cells; and day 32, typical cholangiofibrosis defined by hyperplastic bile ductule-type structures in association with an increased number of metaplastic intestinal glands and progressively more dense fibrotic stroma. Interestingly, at 16 and 32 days the cellular composition of the newly formed metaplastic intestinal glands in liver closely resembled that of the crypts of Lieberkühn in the normal adult rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic characterization of metaplastic intestinal glands and ductular hepatocytes in cholangiofibrotic lesions rapidly induced in the caudate liver lobe of rats treated with furan.

In order to investigate the early cellular changes in liver associated with furan cholangiocarcinogenesis, young adult male Fischer 344 rats were administered furan by gavage once a day, 5 days a wk for 2 to 3 wk at doses ranging from 15 to 60 mg/kg of body weight per day. The most conspicuous feature observed in the liver of animals receiving the higher doses of furan was a rapidly developed cholangiofibrosis characterized by the presence of bile ductular hyperplasia, intestinal metaplasia, and fibrosis. Moreover, this lesion was found to be almost exclusively localized to the caudate liver lobe, which by morphometric analysis was further determined to be largely replaced by cholangiofibrotic tissue. Both the hyperplastic bile ductular epithelial cells and the intestinal-like epithelial cells in these areas selectively exhibited a strongly positive immunohistochemical staining for cytokeratin 19 and were supported by well-developed basement membranes enriched in both laminin and type IV collagen. However, in contrast to the hyperplastic bile ductules, electron microscopy of the metaplastic intestinal glands revealed them to be composed mostly of columnar epithelial cells with well-developed striated borders, less numerous mucin-secreting goblet cells, and occasional neuroendocrine-like cells, thus closely resembling in their cellular composition that of intestinal mucosa. These metaplastic glands also showed a more heterogeneous pattern of staining for both gamma-glutamyl transpeptidase and the placental form of glutathione S-transferase than did the hyperplastic bile ductules. At the 60-mg/kg/day furan dose, cholangiolar-like structures composed of biliary epithelial cells and ductular hepatocytic cells at different stages of morphological differentiation were also observed. Phenotypically, the biliary epithelial and "ductular hepatocytes" of these cholangioles shared a common basement membrane containing laminin and type IV collagen, as well as a luminal plasma membrane gamma-glutamyl transpeptidase. On the other hand, only the biliary epithelial cells of the newly appearing mixed cell cholangioles stained positive for cytokeratin 19. Interestingly, unlike hepatocarcinogen-induced oval cells, alpha-fetoprotein expression was not detected in any of the cell types comprising the furan-induced cholangiofibrotic tissue. These results support a novel in vivo model for investigating cell lineages in the development in liver of intestinal metaplasia, "ductular hepatocytes," and cholangiofibrosis in relation to intrahepatic cholangiocarcinogenesis.

Characterization of rat hyperplastic bile ductular epithelial cells in culture and in vivo.

A novel intrahepatic biliary cell culture/in vivo transplantation system has been developed with an essentially pure population of bile ductular epithelial cells isolated from rat liver 6-12 weeks after bile duct ligation. In primary culture, these cells retain staining strongly for gamma-glutamyltranspeptidase and glutathione S-transferase P. The cytoplasm of cultured bile ductular cells reacts with an anti-laminin antibody, but loses immunoreactivity with a monoclonal anti-cytokeratin 19 antibody. Semiconservative DNA synthesis in the cultured cells was dependent upon the continued presence of 10% fetal calf serum in the medium. Replicating bile ductular cells could be subcultured for a finite number of passages. In addition, freshly isolated bile ductular epithelial cells gave rise to well differentiated bile ductular structures when transplanted into the interscapular fat pads of syngeneic recipient rats.

Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells.

Recent advances made in the isolation, culture, and transplantation of defined populations of intrahepatic biliary epithelial cells and oval cells have permitted direct analysis of the functions, growth properties, and differentiation potential of these respective cell types in their untransformed or transformed states. This review provides a current and comprehensive examination of the various approaches that have been taken to isolate and culture intrahepatic biliary epithelial cells from normal and cholestatic liver and oval cells from preneoplastic liver. Emphasis is placed on comparing the phenotypic features and growth properties of these various biliary cell types in vitro as well as on describing their transplantation into ectopic tissue sites. In addition, the oval cell is evaluated in terms of its potential role as a 'facultative stem cell' during hepatocarcinogenesis.

A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor.

We find that cells infected with wild-type group C human adenoviruses are not killed by exposure to tumor necrosis factor (TNF), but cells infected with adenoviruses that delete the E3 transcription unit are highly sensitive to TNF lysis. Mock-infected cells are resistant to TNF. Thus, adenovirus infection induces cellular susceptibility to lysis by TNF, and a product of E3 protects against lysis by TNF. The E3-dependent resistance to TNF was investigated using virus mutants that delete different segments of E3. Resistance was found to depend on the presence of a 14,700 MW protein, which has only recently been identified and for which there was no known function. Our results support the hypothesis that one of the functions of TNF in vivo is to combat virus infections, and that the 14,700 MW protein evolved in adenovirus to counteract the antiviral effects of TNF.