James O. Price

Reversible G1 Arrest Induced by Inhibition of the Epidermal Growth Factor Receptor Tyrosine Kinase Requires Up-regulation of p27KIP1 Independent of MAPK Activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G1 to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1–10 μm AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1–1 μm AG-1517 abrogated 125I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 μm. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G1 phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G1 arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G1 arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G1. These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G1 arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.

Anti-CD20 treatment depletes B-cells in blood and lymphatic tissue of cynomolgus monkeys

INTRODUCTION Macaque species offer a valuable model for translational allo-transplantation and tolerance studies. Cardiac allograft vasculopathy in Macaca fascicularis is associated with elaboration of anti-donor antibodies. Since T-independent pathways of B cell activation have been described, and anti-B cell strategies have proven to be a fruitful tolerogenic adjunct in rodent and xenogenic models, here we investigate whether an anti-CD20 antibody (rituximab) would be useful to deplete B-cells in a pre-clinical allo-transplantation setting in macaques. METHODS Three cynomolgus macaques which had previously rejected a cardiac allograft and one with concurrent subacute vascular rejection were treated weekly with rituximab 20 mg/kg i.v. for 4 and 2 weeks, respectively. B-cell levels (CD19+ cells) were measured by flow cytometry in peripheral blood, spleen, lymph node and bone marrow cells at various intervals after initiation of treatment. B-cells and plasma cells were also analyzed by immunohistochemistry at necropsy in spleen, lymph node, tonsil and thymus tissue sections. Anti-donor antibody titers were measured by flow cytometry. RESULTS B-cells expressing CD19 were not detectable in the peripheral blood in any animal within 24 h after initial treatment, or over the ensuing month. At necropsy, the germinal centers in spleen and lymph node were completely depleted of CD20+ B-cells in 2 animals, leaving a hypocellular trabecular pattern around preserved plasma cell follicles. Substantial but incomplete depletion of B-cells was demonstrated in the other 2 animals, in each instance immunohistochemical findings in spleen and lymph node exhibiting higher sensitivity for residual B-cells compared to FACS. Anti-donor antibody titers exhibited kinetics similar to untreated animals over this short follow-up. COMMENT Treatment with anti-CD20 very efficiently depletes peripheral and tissue B-cells but not plasma cells in this macaque species. Biopsy of lymph node is necessary and may be sufficient to assess B-cell clearance in secondary lymphoid organs in this model.

THE NOVEL TAXANE ANALOGS, BMS-184476 AND BMS-188797, POTENTIATE THE EFFECTS OF RADIATION THERAPY IN VITRO AND IN VIVO AGAINST HUMAN LUNG CANCER CELLS

PURPOSE To evaluate the novel taxane analogs, BMS-184476 and BMS-188797, as potential radiosensitizers in vitro and in vivo. METHODS AND MATERIALS Human H460 lung cancer cells were incubated with either paclitaxel or a taxane analog and irradiated at various times. Surviving fractions were then determined using a clonogenic assay. Three different schedules were used: (A) 1-h drug incubation with radiation at t = 8 h, (B) 1-h drug incubation with radiation at t = 24 h, (C) 24-h drug incubation with radiation immediately after. Cell cycle redistribution by taxanes alone was measured with propidium iodide and flow cytometry. Percent apoptosis was also measured using 7-aminoactinomycin D (7-AAD) staining with flow cytometry. For in vivo studies, H460 cell xenografts were used in nude mice. Tumors were grown s.c. on the flank and then treated with BMS-184476 (10 mg/kg i.p. injection, Days 0, 2, and 4) and/or radiation (2 Gy/day, Days 0-4). Tumor growth delay was then measured for each treatment group. RESULTS The mean in vitro radiation dose enhancement ratios of BMS-184476, BMS-188797, and paclitaxel were 1.76, 1.49, and 1.31 for Schedules A, B, and C, respectively. Isobologram analysis showed that BMS-184476 was synergistic with radiation using Schedule A. Treatment with taxanes caused an increase in the percentage of G2/M cells at the time of irradiation. The mean fold increases in the %G2/M above control values for all three drugs were 5.6, 2.5, and 1.7 for Schedules A, B, and C, respectively. The combined effects of taxanes plus radiation on the induction of apoptosis were additive for all three drugs. In vivo studies showed that BMS-184476 can enhance the effects of fractionated radiotherapy, with an average enhancement factor of 1.66 obtained from three independent experiments. CONCLUSIONS These results demonstrated that the novel taxane analogs, BMS-184476 and BMS-188797, can enhance the effects of radiation in human lung cancer cells both in vitro and in vivo. These data also support the hypothesis that a G2/M block is involved in the radiosensitization caused by the taxanes.

Reduction of cell cycle progression in human erythroid progenitor cells treated with tumour necrosis factor alpha occurs with reduced CDK6 and is partially reversed by CDK6 transduction

Summary. Tumor necrosis factor α (TNFα) potently inhibits the in vitro growth of highly purified human d‐6 erythroid colony forming cells (ECFC). Unlike the inhibitory effect of TNFα on other cells, including more immature ECFC, this antiproliferative effect of TNFα is not related to apoptosis because the d‐6 cell descendants were morphologically normal, without apoptosis by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labelling assay and without caspase activation by Western blots after TNFα treatment. TNFα did not appear to affect the cell cycle distribution, but the cell cycle duration was significantly longer in TNFα‐treated cells. DNA synthesis was also significantly reduced by TNFα. Studies of various proteins that regulate the cell cycle showed that cyclin‐dependent kinase 6 (CDK6) protein and mRNA levels were concomitantly decreased in the presence of TNFα, suggesting that inhibition of cell growth was related to reduced CDK6. To evaluate this, the CDK6 gene was transferred into ECFC using green fluorescence protein‐retrovirus‐mediated gene transfer. The results showed that the level of cell growth produced by TNFα was increased by 30% when the cells were transfected with CDK6. Therefore, the modification of cell cycle progression in the presence of TNFα through a reduction of CDK6 is an important mechanism in the TNFα inhibition of human ECFC expansion.

Breath-holding spell and macrocytic anaemia in a toddler

A 23-month-old toddler presented with a breath holding episode. She was thriving with normal development and no neurological abnormality. An automated full blood count showed her haemoglobin concentration to be 56 g/l with an MCV of 99 fl [normal range (NR) for age 72–87], an MCH of 33 pg (NR 24–32) and a normal white cell and platelet count. Her serum vitamin B12, red cell folate, thyroid function and liver function tests were normal. She had a markedly raised serum lactate of 4 7 mmol/l without evidence of metabolic acidosis. Further metabolic investigation, which was carried out because of the possibility of a mitochondrial disorder, showed a greatly elevated plasma alanine and plasma proline, at 787 lmol/l (NR 156–547) and 405 lmol/l (NR 59– 369), respectively. Her blood pyruvate was mildly elevated at 0 16 mmol/l (NR < 0 1) whereas her urine metabolic screening was normal. Low faecal pancreatic elastase was suggestive of pancreatic insufficiency. Bone marrow studies showed ring sideroblasts and erythroid cytoplasmic vacuolation (images). Cytogenetic analysis was normal. A diagnosis of Pearson syndrome was confirmed by molecular analysis of bone marrow, which showed a 2-kilobase deletion of mitochondrial DNA. She receives red cell transfusion every 2 months, together with supplements of oral co-enzyme Q10, vitamin C, thiamine and riboflavin. She continues to be well, with normal cardiac function, 9 months after her initial diagnosis. Pearson syndrome is characterized by sideroblastic anaemia with vacuolization of bone marrow precursors and exocrine pancreatic dysfunction. It is caused by sporadic deletions and/or duplications of the mitochondrial DNA, diagnosed by Southern blot analysis of leucocytes, leading to a deficiency in the mitochondrial respiratory chain. Pearson syndrome is often fatal in infancy due to septicaemia, metabolic acidosis or hepatocellular insufficiency.