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Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron.

Messner DJ, Kowdley KV - BMC Gastroenterol (2008)

Bottom Line: Within 1 week of treatment at 200 microM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01).The iron chelator desferoxamine reduced both iron uptake and colony formation.Cells cultured with 200 microM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bastyr University, 14500 Juanita Drive, Kenmore, WA 98028, USA. dmessner@BASTYR.EDU

ABSTRACT

Background: Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI) also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies.

Methods: T51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC) to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot.

Results: T51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 microM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01). FAC alone induced little or no colony formation in soft agar. In contrast, FAC addition to cells previously initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in a concentration dependent increase in colony formation. This was first detected at 12 weeks of FAC treatment and increased at longer times. At 16 weeks, colony formation increased more than 10 fold in cells treated with 200 microM FAC (p < 0.001). The iron chelator desferoxamine reduced both iron uptake and colony formation. Cells cultured with 200 microM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.

Conclusion: These results establish NTBI as a tumor promoter in T51B rat liver epithelial cells. Changes in cyclin proteins suggest cell cycle disregulation contributes to tumor promotion by NTBI in this liver cell model.

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Iron alters the cell cycle distribution of T51B cells. Proliferating cells were treated with the indicated concentrations of FAC (in μM) for 5 days and processed for western blots using antibodies specific for cyclin D1, cyclin E, cyclin A, cyclin B1, and GAPDH as gel loading control. All panels are from a single experiment and are representative of results obtained at least 4 times.
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Figure 4: Iron alters the cell cycle distribution of T51B cells. Proliferating cells were treated with the indicated concentrations of FAC (in μM) for 5 days and processed for western blots using antibodies specific for cyclin D1, cyclin E, cyclin A, cyclin B1, and GAPDH as gel loading control. All panels are from a single experiment and are representative of results obtained at least 4 times.

Mentions: At tumor promoting concentrations, FAC did not increase T51B cell proliferation, but rather inhibited it slightly (Figure 2A). The levels of cyclin proteins should inform on the nature of this effect and may suggest a basis for tumor promotion [47]. Figure 4 shows the effect of FAC on the levels of cyclin proteins in T51B cells. Modest reductions in a G1-phase cyclin (D1) and the S-phase cyclin (A) correlated well with the slight decrease in proliferation seen in cells exposed to tumor promoting concentrations of FAC (200 μM). And, a more complete loss from cells exposed to 500 μM FAC correlated well with the proliferative block seen at that concentration. There was a concomitant decrease in junB levels and AP-1 activity (data not shown) that may partially explain these decreases. Other cyclins not primarily regulated by AP-1, including a G1 cyclin (E) and the M-phase cyclin (B1), did not decrease in FAC-treated cells. Cyclin E levels remained unchanged, while cyclin B1 levels increased significantly (Figure 4). The increase in cyclin B1 was not seen in cells treated with ammonium citrate controls or when the bulk of the iron was chelated by dfo (not shown).


Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron.

Messner DJ, Kowdley KV - BMC Gastroenterol (2008)

Iron alters the cell cycle distribution of T51B cells. Proliferating cells were treated with the indicated concentrations of FAC (in μM) for 5 days and processed for western blots using antibodies specific for cyclin D1, cyclin E, cyclin A, cyclin B1, and GAPDH as gel loading control. All panels are from a single experiment and are representative of results obtained at least 4 times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275280&req=5

Figure 4: Iron alters the cell cycle distribution of T51B cells. Proliferating cells were treated with the indicated concentrations of FAC (in μM) for 5 days and processed for western blots using antibodies specific for cyclin D1, cyclin E, cyclin A, cyclin B1, and GAPDH as gel loading control. All panels are from a single experiment and are representative of results obtained at least 4 times.
Mentions: At tumor promoting concentrations, FAC did not increase T51B cell proliferation, but rather inhibited it slightly (Figure 2A). The levels of cyclin proteins should inform on the nature of this effect and may suggest a basis for tumor promotion [47]. Figure 4 shows the effect of FAC on the levels of cyclin proteins in T51B cells. Modest reductions in a G1-phase cyclin (D1) and the S-phase cyclin (A) correlated well with the slight decrease in proliferation seen in cells exposed to tumor promoting concentrations of FAC (200 μM). And, a more complete loss from cells exposed to 500 μM FAC correlated well with the proliferative block seen at that concentration. There was a concomitant decrease in junB levels and AP-1 activity (data not shown) that may partially explain these decreases. Other cyclins not primarily regulated by AP-1, including a G1 cyclin (E) and the M-phase cyclin (B1), did not decrease in FAC-treated cells. Cyclin E levels remained unchanged, while cyclin B1 levels increased significantly (Figure 4). The increase in cyclin B1 was not seen in cells treated with ammonium citrate controls or when the bulk of the iron was chelated by dfo (not shown).

Bottom Line: Within 1 week of treatment at 200 microM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01).The iron chelator desferoxamine reduced both iron uptake and colony formation.Cells cultured with 200 microM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bastyr University, 14500 Juanita Drive, Kenmore, WA 98028, USA. dmessner@BASTYR.EDU

ABSTRACT

Background: Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI) also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies.

Methods: T51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC) to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot.

Results: T51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 microM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01). FAC alone induced little or no colony formation in soft agar. In contrast, FAC addition to cells previously initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in a concentration dependent increase in colony formation. This was first detected at 12 weeks of FAC treatment and increased at longer times. At 16 weeks, colony formation increased more than 10 fold in cells treated with 200 microM FAC (p < 0.001). The iron chelator desferoxamine reduced both iron uptake and colony formation. Cells cultured with 200 microM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1.

Conclusion: These results establish NTBI as a tumor promoter in T51B rat liver epithelial cells. Changes in cyclin proteins suggest cell cycle disregulation contributes to tumor promotion by NTBI in this liver cell model.

Show MeSH
Related in: MedlinePlus