The effects of 3′,5′ adenosine monophosphate on the proliferation of reuber H35 rat hepatoma cells in vitro

Summary 3′,5′ Adenosine monophosphate (cAMP) inhibits the proliferation of Reuber H35 rat hepatoma cells at concentrations higher than 10⁻⁵ M. This inhibitory effect can be demonstrated both in exponentially growing monolayer cultures and in single cell clonal growth. The inhibition of the cell proliferation is due to both a short term (division delay) and a long term effect (cytotoxicity). The short term effect seems to be due to cAMP itself as it is potentiated by the phosphodiesterase inhibitor 1-methyl,3-isobutyl xanthine (MIX). The long term effect probably is due to degradation products of the cyclic nucleotide. It is shown by a combination of time lapse cinematography, autoradiography, and flow cytofluorometry that the division delay is due to a prolongation of the S phase with no apparent changes in the duration of the G1 phase. The possible causes of this prolongation of the S phase by cAMP are discussed. This investigation was supported by the Netherlands Cancer Society (Koningin Wilhelmina Fonds).     

Drug screening assay based on the interaction of intact Keap1 and Nrf2 proteins in cancer cells

Background

The Nrf2–Keap1 interaction is the major regulatory pathway for cytoprotective responses against oxidative and electrophilic stresses. Keap1, a substrate protein of a Cul3-dependent E3 ubiquitin ligase complex, is a negative regulator of Nrf2. The use of chemicals to regulate the interaction between Keap1 and Nrf2 has been proposed as a strategy for the chemoprevention of degenerative diseases and cancers.

Loss of FANCC function is associated with failure to inhibit late firing replication origins after DNA cross-linking

Fanconi anemia (FA) cells are abnormally sensitive to DNA cross-linking agents with increased levels of apoptosis and chromosomal instability. Defects in eight FA complementation groups inhibit monoubiquitination of FANCD2, and subsequent recruitment of FANCD2 to DNA damage and S-phase-associated nuclear foci. The specific functional defect in repair or response to DNA damage in FA cells remains unknown. Damage-resistant DNA synthesis is present 2.5-5 h after cross-linker treatment of FANCC, FANCA and FANCD2-deficient cells. Analysis of the size distribution of labeled DNA replication strands revealed that diepoxybutane treatment suppressed labeling of early but not late-firing replicons in FANCC-deficient cells. In contrast, normal responses to ionizing radiation were observed in FANCC-deficient cells. Absence of this late S-phase response in FANCC-deficient cells leads to activation of secondary checkpoint responses.     

H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia

Immunocytochemistry using α-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases α and δ, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1^S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2^T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX agregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.     

Assessment of cell proliferation by Ki-67 staining and flow cytometry in fine needle aspirates (FNAs) of reactive lymphadenitis and non-Hodgkin's lymphomas

This study was undertaken to assess cell proliferation in FNAs from a series of 57 non-Hodgkin's lymphomas (NHL) and 11 cases of reactive lymphadenitis using Ki-67 staining and flow cytometry (FCM). The results were compared and correlated to the cytomorphological subgrouping according to Kiel classification. The mean percentages of Ki-67 positivity were 16.6% and 61.1% for low and high grade lymphomas, respectively (P < 0.001). The mean S-phase fraction (SPF) determined by FCM was 4.61% for low grade and 12.9% for high grade lymphomas (P < 0.001). The figures for Ki-67 positivity and S-phase fraction in reactive lymphadenitis were 16.8% and 40%, respectively. We observed a strong correlation in low grade lymphomas between Ki-67 and SPF. A good correlation was also found in reactive lymphadenitis. In high grade lymphomas, however, with highly scattered Ki-67 and S-phase values, this correlation was lost. In some cases this discrepancy can be explained by a rich admixture of non-neoplastic, non-proliferating cells in aspirates from diploid tumours. In addition, the existence of a minor aneuploid tumour cell population of high proliferation such as that in Ki-1 lymphomas will not be accurately analysed by FCM but is easily assessed by Ki-67 staining. However, the main reason seems to be a high variability between the fraction of cells in S-phase and the total number of cells in G1, S and G2 in individual tumours.     

Immediate-Early Gene Expression during Regenerative and Mitogen-Induced Liver Growth in the Rat

The nongenotoxic carcinogens phenobarbitone (PB) and methyl clofenapate (MCP) and the hepatomitogen pregnenolone 16α carbonitrile (PCN) are direct inducers of hepatic S -phase in rats, whereas the S -phase seen after partial hepatectomy is regenerative. We have investigated S -phase and immediate-early gene expression (c-myc and c-jun) in rat liver following these treatments to study the differences in gene expression associated with direct vs. regenerative responses. Both partial hepatectomy (one- and two-thirds) and mitogen treatment caused an increase in hepatic S -phase that peaked around 36 hours. Two-thirds partial hepatectomy caused the greatest increase in S -phase followed by one-third partial hepatectomy, then the mitogens PCN, MCP, and PB in that order. This order of response was also seen with c-jun and to a lesser degree with c-myc expression, suggesting that immediate-early gene expression might be linked not only to regenerative S -phase but also to direct mitogen-induced responses. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 79–82, 1998     

Utility and sensitivity of anti BrdU antibodies in assessing S-phase cells compared to autoradiography

A rapid and convenient method for estimating S-phase cells in a population was developed which detects bromodeoxyuridine (BrdU) incorporation into DNA by means of monoclonal anti-BrdU antibodies. This immunofluorescence technique (RPMB technique) was compared to autoradiographic (ARG) detection of tritiated thymidine (3HTdr) grains incorporated into the DNA. Using incubation periods for BrdU and 3HTdr ranging from one minute to one hour and detecting their incorporation by ARG and RPMB techniques, it became apparent that the RPMB technique was far more sensitive than ARG in addition to being extremely easy to perform. Some possible utilities of the RPMB technique are discussed.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.