Thoralf Christoffersen

Phase III Trial of Cetuximab With Continuous or Intermittent Fluorouracil, Leucovorin, and Oxaliplatin (Nordic FLOX) Versus FLOX Alone in First-Line Treatment of Metastatic Colorectal Cancer: The NORDIC-VII Study

PURPOSE The NORDIC-VII multicenter phase III trial investigated the efficacy of cetuximab when added to bolus fluorouracil/folinic acid and oxaliplatin (Nordic FLOX), administered continuously or intermittently, in previously untreated metastatic colorectal cancer (mCRC). The influence of KRAS mutation status on treatment outcome was also investigated. PATIENTS AND METHODS Patients were randomly assigned to receive either standard Nordic FLOX (arm A), cetuximab and FLOX (arm B), or cetuximab combined with intermittent FLOX (arm C). Primary end point was progression-free survival (PFS). Overall survival (OS), response rate, R0 resection rate, and safety were secondary end points. RESULTS Of the 571 patients randomly assigned, 566 were evaluable in intention-to-treat (ITT) analyses. KRAS and BRAF mutation analyses were obtained in 498 (88%) and 457 patients (81%), respectively. KRAS mutations were present in 39% of the tumors; 12% of tumors had BRAF mutations. The presence of BRAF mutations was a strong negative prognostic factor. In the ITT population, median PFS was 7.9, 8.3, and 7.3 months for the three arms, respectively (not significantly different). OS was almost identical for the three groups (20.4, 19.7, 20.3 months, respectively), and confirmed response rates were 41%, 49%, and 47%, respectively. In patients with KRAS wild-type tumors, cetuximab did not provide any additional benefit compared with FLOX alone. In patients with KRAS mutations, no significant difference was detected, although a trend toward improved PFS was observed in arm B. The regimens were well tolerated. CONCLUSION Cetuximab did not add significant benefit to the Nordic FLOX regimen in first-line treatment of mCRC.

ORAL CONTRACEPTIVE USE AND BREAST CANCER IN YOUNG WOMEN: A Joint National Case-control Study in Sweden and Norway

The possible association between oral contraceptive (OC) use and the risk of breast cancer developing before the age of 45 was investigated by means of a population based case-control study in Sweden and Norway. Information was obtained by personal interview from 422 (89.2%) of all eligible patients with a newly diagnosed breast cancer from May, 1984, to May, 1985, and from 722 (80.6%) of all contacted age-matched controls. A multivariate analysis, which accounted for several possible confounding factors, revealed a significant (p = 0.03) association between total duration of OC use and breast cancer risk. The relative risk (RR) of breast cancer after 12 or more years of OC use was 2.2 (1.2-4.0). OC use for more than 7 years before first full-term pregnancy entailed an increased breast cancer risk (RR = 2.0 [1.0-4.2]) which was of borderline significance. When total duration of use was considered, the risk of breast cancer was virtually unrelated to age at first OC use and latency from first use. The results suggest that long-term use of OCs may increase the risk of breast cancer in young women.

Development of cyclic AMP metabolism in rat liver a correlative study of tissue levels of cyclic AMP, accumulation of cyclic AMP in slices, adenylate cyclase activity and cyclic nucleotide phosphodiesterase activity

Abstract Developmental alterations in the levels of cyclic AMP, adenylate cyclase activity, phosphodiesterase activity and accumulation of cyclic AMP in slices during incubation in vitro were studied in male rat liver. 1. 1. At 1 week before birth adenylate cyclase activity was present. At this stage the enzyme was activated by fluoride, but not by adrenalin or glucagon. At 2 days before birth both adrenalin and glucagon stimulated the adenylate cyclase. 2. 2. During the perinatal period the tissue levels of cyclic AMP, the adenylate cyclase activity and the production of cyclic AMP in slices increased rapidly, reaching maximal values towards the end of the first postnatal week. This peak coincided with a maximal phosphodiesterase activity, indicating an increased cyclic AMP turnover in neonatal liver. 3. 3. When the animals grew older the tissue levels and the rate of synthesis and breakdown of cyclic AMP declined. In addition, the response to adrenalin and glucagon developed differently with increasing age: the stimulatory effect of adrenalin on adenylate cyclase activity and cyclic AMP formation in slices gradually disappeared, whereas response to glucagon persisted.

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.

Response to transforming growth factor α (TGFα) and epidermal growth factor (EGF) in hepatocytes: Lower EGF receptor affinity of TGFα is associated with more sustained activation of p42/p44 mitogen-activated protein kinase and greater efficacy in stimulation of DNA synthesis

The epidermal growth factor (EGF) receptor mediates the effects of both EGF and transforming growth factor α (TGFα). Recent data suggested that EGF acts as a partial agonist/antagonist in hepatocytes, TGFα exerting a larger maximal stimulation of DNA synthesis than EGF. To further study the mechanisms involved in mediating the different effects of EGF and TGFα, we have examined receptor binding of the two growth factors and their action on the p42/p44 mitogen‐activated protein (MAP) kinase activity in hepatocytes. Single‐ligand concentration curves and competition experiments showed that the binding affinity to a common population of surface binding sites was about 20‐fold lower for TGFα than for EGF. MAP kinase activity responded to EGF and TGFα with different kinetics. While the two agents produced almost identical acute (5 min) stimulation (peak about fivefold), TGFα produced a more sustained MAP kinase activity than EGF. The difference between EGF and TGFα was still detectable 24 h after growth factor addition. The results show that in hepatocytes a lower receptor affinity of TGFα, as compared to EGF, is associated with a more sustained activation of the MAP kinase and a greater efficacy in the stimulation of DNA synthesis. This suggests that differential interaction of these two agents with the EGF receptor results in differences in the downstream events elicited at a given level of receptor occupancy. The data also are compatible with a role of a prolonged MAP kinase activity in the mitogenic effects of EGF and TGFα. J. Cell. Physiol. 175:10–18, 1998.

Increased effect of adrenaline on cyclic AMP formation and positive β‐adrenergic modulation of DNA‐synthesis in regenerating hepatocytes

Hormonal factors are impo~~t regulators of eukaryotic cell proliferation [l-4]. Changes in the way cells respond to humoral agents may, therefore, alter their growth properties. The initiation of DNAsynthesis and proliferation of liver cells occurring after partial hepatectomy [5], seems to require certain growth factors, including insulin and possibly epidermal growth factor (EGF) [6]. Moreover, several observations suggest a stimulatory role of cyclic AMP. Hepatic DNA-synthesis can be induced in vivo by infusion of triiodothyronine, heparin and amino acids in combination with glucagon [7] or ~butyryl cyclic AMP [8 1. In primary cultures of adult rat hepatocytes, addition of insulin, glucagon, EGF and dexamethasone stimulates DNA-synthesis 191. After partial hepatectomy elevation of the cyclic AMP level precedes [lo] and may be required for [ 11) the onset of DNA-replication, It is not clear how this rise in cyclic AMP is produced. We here show that after partial hepatectomy the liver adenylate cyclase activity and the accumulation of cyclic AMP in isolated hepatocytes become more responsive to adrenaline. Fu~hermore, an indication that adrenergic activation may represent a growth stimulus for hepatocytes, is provided by the demonstration that the fi-adrenergic agent isoprenaline in low concentrations (10-10-10” mol/l), enhanced DNA-synthesis in these cells in culture, It may be noteworthy that increased adrenaline responsiveness has been found also in growing immature [ 121 and premalignant [13,14] liver.

Hepatic adenyl cyclase: Alterations in hormone response during treatment with a chemical carcinogen

Abstract 1. 1. The response of liver adenyl cyclase to adrenalin increased markedly after treatment of adult rats with the carcinogen 2-acetylaminofluorence for a few weeks. This change, which made the hormone stimulation pattern of the enzyme resemble that of immature liver, occurred before histological signs of malignancy appeared. 2. 2. Fully developed hepatocarcinomas had high basal adenyl cyclase activity, but small responses to hormones.

Activation of p42/p44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2α in hepatocytes is sustained, and like the effect of epidermal growth factor, mediated through pertussis toxin-sensitive mechanisms

Several agents that act through G‐protein‐coupled receptors and also stimulate phosphoinositide‐specific phospholipase C (PI‐PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2α, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen‐activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in‐gel assays. All these agonists induced a peak activation (two to threefold increase in MBP‐phosphorylation) at 3–5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI‐PLC, implicating a Gq‐dependent pathway, the elevations of the concentration of inositol (1,4,5)‐trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2α. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G‐protein‐coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi‐dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G‐protein‐coupled receptors as well as the EGF receptor. J. Cell. Physiol. 175:348–358, 1998.

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes.

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR‐independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2‐stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor‐sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF‐stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2‐treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part Gi protein‐mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways. J. Cell. Physiol. 214: 371–380, 2008.

Glucagon control of cyclic AMP accumulation in isolated intact rat liver parenchymal cells in vitro

Cyclic AMP levels were measured in suspensions of isolated rat liver parenchymal cells during incubation in vitro. Glucagon caused a rapid elevation of cyclic AMP content. With 1.4·10−6 M (5 μg/ml) of the hormone the levels increased about 10-fold during the first minute, thereafter the elevation was less rapid. Maximal values were reached at 5–10 min. Theophylline slightly increased the basal cyclic AMP levels, and markedly augmented the response to glucagon. Teh major part of the cyclic AMP was located within cells, but a siginificant fraction was present in the incubation medium, and the relative amount present extracellularly increased with incubation time. Significant elevation of the cyclic AMP levels was produced by glucagon ⩾1.4·10−10M, and half-maximal stimulation occured at about 2·10−9 M. The initial rate of cyclic AMP accumulation was such more rapid in the parenchymal cells than in liver slices, and the maximal levels obtained were about 3 times higher (comparisons based on the finding that 1 mg liver tissue contains about 105 parenchymal cells). It is concluded that preparations of parenchymal liver cells are useful in the study of glucagon actions on liver tissue.