p57Kip2 is an unrecognized DNA damage response effector molecule that functions in tumor suppression and chemoresistance.

The DNA damage response (DDR) helps to maintain genome integrity, suppress tumorigenesis and mediate the radiotherapeutic and chemotherapeutic effects on cancer. Here we report that p57Kip2, a cyclin-dependent kinase (CDK) inhibitor implicated in the development of tumor-prone Beckwith-Wiedemann syndrome, is an effector molecule of the DNA-damage response. Genotoxic stress induces p57Kip2 expression via the bone morphogenetic protein-Smad1 and Atm-p38MAPK-Atf2 pathways in p53-proficient or -deficient cells and requires the Smad1-Atf2 complex that facilitates their recruitment to the p57Kip2 promoter. Elevated p57Kip2 induces G1/S phase cell cycle arrest but inhibits cell death in response to DNA damage and acts in parallel with p53 to suppress cell transformation and tumor formation. p57Kip2 is also upregulated in stage I and II clinical rectal tumor samples, likely due to genome instability of precancerous and/or early cancer cells. Targeting p57Kip2 in primary rectal cancer cells and tumor models resulted in increased sensitivity to doxorubicin, suggesting that p57Kip2 has a role in chemoresistance, which is consistent with its pro-survival function. These findings place p57Kip2 in DDR and uncover molecular mechanisms by which p57Kip2 suppresses tumorigenesis and causes chemoresistance.

Modifications in the carcinogen-metabolizing capacity of mouse liver treated with N-nitroso compounds.

One tenth of the LD50 as a single dose of various N-nitroso compounds (N-nitrosodimethylamine; NDMA, N-nitrosodiethylamine; NDEA, N-nitrosoethylpropylamine; NEPA, N-nitrosodipropylamine; NDPA, N-nitrosomethylethylamine; NMEA, N-nitroso-methylbutylamine; NMBA and N-nitrosoethylbutylamine; NEBA) was administrated into male mice. This dose markedly increased the hepatic contents of cytochrome P450 and cytochrome b5 and activities of NADPH-cytochrome c reductase and aryl hydrocarbon hydroxylase (AHH). The highest increase in the activity of cytochrome P450 (+142% relative to the control value) was shown in animals treated with either N-nitrosoethylpropylamine or N-nitrosodiethylamine. On the other hand, the lowest increase in the activity (+16%) was revealed in animals treated with N-nitrosodimethylamine (not significant compared to the control value). Cytochrome b5 content was increased by 190% of the control value in mice treated with N-nitrosomethylbutylamine, while N-nitrosodibutylamine induced the lowest increase (+20%). The maximum increase (+182%) in the activity of aryl hydrocarbon hydroxylase was shown in animals which received N-nitrosomethylbutylamine, while the lowest increase (+23%) in animals which received N-nitrosodiethylamine. The activity of hepatic AHH was also increased above the control value in animals treated with NDMA, NEBA NDPA, NMEA and NDBA by 138, 98, 90, 89 and 69%, respectively. Identically, NADPH-cytochrome c reductase activity was increased in animals which received NEPA, NMBA, NDMA, NMEA, NDPA, NEBA and NDEA by 202, 150, 110, 95, 94, 77 and 37%, respectively.

Altered expression of cytochrome P450 mRNA during chemical-induced hepatocarcinogenesis and following partial hepatectomy.

Levels of various cytochrome P450 proteins have been reported to be decreased to varying degrees in chemically induced hepatocyte nodules and following partial hepatectomy (PH). By screening a rat liver lambda ZAP cDNA expression library with antibodies raised against a partially purified preparation of cytochrome P450 isolated from untreated male Fischer 344 rats, we have isolated a 1.1-kb cDNA. This cDNA was sequenced for 139 bases from the 5' end of the sense strand and comparison of the resulting sequence with the sequences in Gene Man DNA data bank revealed 95% homology of the sequenced portion with male-specific rat cytochrome P450 (M-1, CYP IIC11). The 32P-labeled cDNA was used as a hybridization probe on RNA blots (Northern blots) prepared with total RNA from rat livers obtained post PH, from aflatoxin B1(AFB1)-induced rat liver tumors and from rat liver nodules induced with a combination of diethylnitrosamine/acetylaminofluorene/PH (DEN/AFF/PH). At 36 and 72 hr post PH, the mRNA level was decreased by > 93%. Relative to the corresponding control livers, the mRNA level was also decreased by 97% in the liver nodules and by 57% in AFB1-induced liver tumors. The RNA blots derived from the liver nodules and AFB1-induced liver tumors were also probed with a cDNA probe (R17) that recognizes other cytochromes P450 (CYP IIB1/CYP IIB2). The mRNA corresponding to CYP IIB1/CYP IIB2 was also depressed 92% in the nodules and 65% in the tumors. These results clearly indicate that the depression of both CYP IIC11 and IIB1/IIB2 in the hepatic nodules and the tumors is related to the inhibition of transcription and/or enhanced degradation of the mRNA.

In vitro effect of aflatoxin B1 on the transcriptional activity of DNA template, chromatin and soluble DNA-dependent RNA polymerases in buffalo liver.

The effect of aflatoxin B1 on the DNA template and DNA-dependent RNA polymerases in buffalo liver was studied. Aflatoxin B1 inhibited both Mg2+- and Mn2+-activated RNA polymerases in a dose-dependent manner. At 10 micrograms the inhibition of both enzymes was almost complete. The inhibitory effect on the solubilized enzymes was higher than the chromatin-bound, suggesting a direct effect at the enzyme level. On the other hand, incubating DNA or deoxyribonucleoprotein (DNP) with 2 micrograms aflatoxin reduces its transcriptional capacity with a greater effect on the Mg2+-activated RNA polymerase than the Mn2+-activated enzyme. These results suggest that aflatoxin B1 inhibits in vitro transcription in buffalo liver at both enzyme and template levels.

Isolation, purification and characterization of enzyme(s) responsible for conversion of sterigmatocystin to aflatoxin B1.

The cell-free extract prepared from Aspergillus flavus ATCC 5517/A 228 showed activity in converting sterigmatocystin to aflatoxin B1. The extract was purified on Ultrogel AcA-54 and resulted in ten protein peaks, one of which (peak VI) showed activity in sterigmatocystin conversion. The protein in this peak gave one protein band using polyacrylamide gel (PAG)-disc electrophoresis. For further purification, protein(s) in peak VI were applied on DEAE-Sephadex A-50 and two protein peaks were detected. Only one peak showed enzyme activity which showed homogeneity as one band on PAGE and sodium dodecyl sulphate (SDS)-PAGE. The optimum temperature for the enzyme activity was 28 degrees C and the optimum pH was 8. The maximum conversion resulted from the action of 0.6 mg enzyme protein on 48 X 10(-8) mol sterigmatocystin. Zn2+, Co2+ and Mn2+ enhanced the enzyme activity, while ethylenediaminetetraacetic acid, parahydroxymercuric benzoate and phenylmethylsulphonic fluoride inhibited the enzyme activity in a dose-dependent manner. Amino-acid analysis showed the presence of 22 amino acids, three of which are unknown. The enzyme has a molecular weight of 64,000 daltons (by gel filtration) and 70,000 daltons (by SDS-PAGE).