scholarly journals Design and Mechanism of Action of a New Prototype of Combi-Molecule “Programed” to Release Bioactive Species at a pH Range Akin to That of the Tumor Microenvironment

2021 ◽  
Vol 14 (2) ◽  
pp. 160
Author(s):  
Anne-Laure Larroque-Lombard ◽  
Etienne Chatelut ◽  
Jean-Pierre Delord ◽  
Diane-Charlotte Imbs ◽  
Philippe Rochaix ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Xenograft Model ◽  
Mek Inhibitor ◽  
Cytotoxic Agents ◽  
Growth Delay ◽  
Novel Approach ◽  
Mitogen Activated Protein ◽  
Ph Range

The clinical use of cytotoxic agents is plagued by systemic toxicity. We report a novel approach that seeks to design a “combi-molecule” to behave as an alkylating agent on its own and to undergo acid-catalyzed conversion to two bioactive species at a pH range akin to that of a tumor microenvironment: an AL530 prototype was synthesized and we studied its ability to release a chlorambucil analogue (CBL-A) plus a potent mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor (PD98059) at different pHs in buffered solutions, plasma and tumors. Its potency was compared in vitro with CBL+PD98059 (SRB assay) and in vivo in a xenograft model. Its target modulation was studied by western blotting and immunohistochemistry. AL530 released PD98059+CBL-A at mild acidic pH and in vitro was fivefold more potent than CBL and three-to-fivefold more potent than CBL+PD98059. In vivo it released high levels of PD98059 in tumors with a tumor/plasma ratio of five. It induced γ-H2AX phosphorylation and blocked pErk1,2, indirectly indicating its ability to damage DNA and modulate MEK. It induced significant tumor delay and less toxicity at unachievable doses for CBL and CBL+PD98059. We demonstrated the feasibility of a pH-labile combi-molecule capable of delivering high MEK inhibitor concentration in tumors, damaging DNA therein, and inducing tumor growth delay.

scholarly journals Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

2021 ◽  
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Growth Delay ◽  
Hsp90 Inhibitors ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored.  Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hiroshi Ikeda ◽  
Teru Hideshima ◽  
Robert J. Lutz ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Growth Delay ◽  
Selective Cytotoxicity ◽  
Bystander Killing

Abstract CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

OM-RCA-01, an FGFR1 specific humanized antibody for the treatment of renal cell carcinoma (RCC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 3070-3070
Author(s):  
Ilya Tsimafeyeu ◽  
Elina Zaveleva ◽  
Walter Low
Keyword(s):  
Renal Carcinoma ◽  
Xenograft Model ◽  
Body Weight Loss ◽  
In Vitro Study ◽  
Growth Delay ◽  
Fgf Receptor ◽  
Specific Igg ◽  
Set Up

3070 Background: Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for the treatment of metastatic RCC. We investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody with high affinity (Kd of 1.59 nM), in RCC. Methods: To assess the effect of anti-FGFR1 antibody on FGF-mediated signaling, the human renal carcinoma Caki-1 FGFR1-expressing cells were dosed with OM-RCA-01 at 100, 10, and 1 mcg/ml. Control wells were left untreated. Three hours after dosing, bFGF was added at a concentration of 50 ng/ml. Additional control wells were treated with OM-RCA-01 without FGF-stimulation. Cell growth inhibition was determined using Promega’s Cell Titer-Glo assay. CR female NCr nu/nu mice were set up with 1 mm3 Caki-1 tumor fragments sc in flank. Tumor sizes were measured in a blind fashion twice a week with a vernier caliper. Mice with established tumors were randomly divided into vehicle, non-specific IgG or OM-RCA-01 groups per 10 animals in group. Endpoint was significant differences in tumor growth delay. Results: In vitro study showed that bFGF increased proliferation of the human FGFR1-expressing renal carcinoma cells (p=0.011). OM-RCA-01 antibody significantly inhibits FGF-triggered cell proliferation in comparison with control. In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors (p=0.006) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses (p=0.917). Administration of 10 mg/kg antibody for up to 35 days resulted in minimal body weight loss and no observations of gross toxicity were made. Conclusions: Targeting FGFR1 blocks FGF/FGFR1 pathway in RCC. Monoclonal antibody OM-RCA-01 has significant early anti-tumor efficacy in Caki-1 xenograft model. Isolated blocking of FGFR1 by low-dose antibody could be safe and effective.

Apatinib to enhance chemosensitivity of gastric cancer to paciltaxel and 5-fluorouracil.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15545-e15545
Author(s):  
Xiangdong Cheng ◽  
Zhiyuan Xu ◽  
Jiahui Chen ◽  
Chunli Zhang ◽  
Jianfa Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Late Stage ◽  
Zebrafish Embryo ◽  
Xenograft Model ◽  
Cytotoxic Agents ◽  
Pathological Examination ◽  
Conversion Surgery ◽  
Synergistic Interactions

e15545 Background: Patients (pts) with late-stage gastric cancer (GC) have a poor prognosis. Targeted agent combined with chemotherapy is expected to yield clinical benefits. Apatinib, a novel tyrosine kinase inhibitor targeting VEGFR-2, improves outcomes in patients with metastatic GC as a third line of treatment. Hence, we aimed to assess the efficacy and safety of apatinib plus chemotherapy in vivo and in vitro. Methods: The MGC803 cell viability was assessed by CCK-8 assay, and the interactions between apatinib and conventional cytotoxic agents revealed by combined index (CI) values were calculated using Calcusyn 2.0 software. We also used a zebrafish embryo xenograft model to validate the synergistic interactions. Furthermore, 4 pts with late-stage GC were enrolled to receive paclitaxel (PTX)/S1 chemotherapy plus apatinib in conversion surgery. Apatinib was administered 500 mg once a day continuously, PTX 130 mg/m2 was given on day 1, and S-1 was administered at 80 mg/m2for 14 consecutive days, followed by 7 days of rest. Treatment was administered for 3-5 cycles, but the last cycle did not include apatinib. Results: Apatinib showed synergistic interactions with both PTX and 5-Fu in vivo (CIs < 1). The zebrafish embryo xenograft model also demonstrated that addition of 0.25 µg/mL apatinib significantly enhanced the tumor growth inhibition effects of 25 (38.39% vs. 11.77%, P < 0.001) and 50 ng/fish (43.58% vs. 17.88%, P < 0.05) 5-Fu, as well as those of 0.75 ng/fish (53.62% vs. 35.22%, P < 0.001) and 1.5 ng/fish (59.71% vs.46.73%, P < 0.01) PTX. Apatinib plus S1/paclitaxel chemotherapy was well tolerable before surgery. Objective response to preoperative SPA treatment was achieved in all pts. No posteroperative bleeding event or wound-healing complication was observed. No postoperative mortality occurred and morbidity was encountered. Pathological examination showed that all pts had grade Ib pathological response. Conclusions: The experimental data suggested that apatinib improves the efficacy of PTX and 5-Fu both in vitro and in vivo. Clinical evidence showed that combination of PTX/S1 chemotherapy with apatinib has promising efficacy and acceptable safety profile in late-stage GC, especially in the conversion surgery.

scholarly journals MEK-inhibitor-mediated rescue of skeletal myopathy caused by activating Hras mutation in a Costello syndrome mouse model

2021 ◽  
Author(s):  
William E. Tidyman ◽  
Alice F. Goodwin ◽  
Yoshiko Maeda ◽  
Ophir D. Klein ◽  
Katherine A. Rauen
Keyword(s):  
Mouse Model ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Costello Syndrome ◽  
Skeletal Myopathy ◽  
Mitogen Activated Protein

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes due to mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due in part to an inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction of myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction of p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS.

scholarly journals Nicotine Induces IL-8 Secretion from Pancreatic Cancer Stroma and Worsens Cancer-Induced Cachexia

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 329
Author(s):  
Patrick W. Underwood ◽  
Dong Yu Zhang ◽  
Miles E. Cameron ◽  
Michael H. Gerber ◽  
Daniel Delitto ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Microenvironment ◽  
Cancer Patient ◽  
Xenograft Model ◽  
Pancreatic Cancer Patient ◽  
Nicotine Treatment ◽  
Pancreatic Cancer Cells ◽  
Cancer Tumor

Smoking is highly associated with pancreatic cancer. Nicotine, the addictive component of tobacco, is involved in pancreatic cancer tumorigenesis, metastasis, and chemoresistance. This work aimed to describe the role of nicotine within the pancreatic cancer tumor microenvironment. Nicotine treatment was used in vitro to assess its effect on tumor-associated stromal cells and pancreatic cancer cells. Nicotine treatment was then used in a pancreatic cancer patient-derived xenograft model to study the effects in vivo. Nicotine induced secretion of interleukin 8 (IL-8) by tumor-associated stroma cells in an extracellular signal-regulated kinase (ERK)-dependent fashion. The secreted IL-8 and nicotine acted on the pancreatic cancer cell, resulting in upregulation of IL-8 receptor. Nicotine treatment of mice bearing pancreatic cancer patient-derived xenografts had significantly increased tumor mass, increased tumor-free weight loss, and decreased muscle mass. These represent important pathways through which nicotine acts within the tumor microenvironment and worsens pancreatic cancer-induced cachexia, potentially representing future therapeutic targets.

Cancer nanomedicine developed from total human serum: a novel approach for making personalized nanomedicine

Nanomedicine ◽  
2021 ◽  
Author(s):  
Jyotsna Thayath ◽  
Keechilat Pavithran ◽  
Shantikumar V Nair ◽  
Manzoor Koyakutty
Keyword(s):  
Xenograft Model ◽  
Total Serum ◽  
Total Blood ◽  
Pancreatic Cell ◽  
Novel Approach ◽  
Cancer Nanomedicine ◽  
Unique Method ◽  
Average Size

Aim: To develop a method for making total serum nanoparticles (TSN) loaded with cytotoxic chemodrugs for cancer therapy. Materials & methods: TSN loaded with paclitaxel (PTX) or piperlongumine (PL) were prepared using high-pressure homogenization and tested for immunogenicity in healthy animals and antitumor properties in pancreatic cancer xenograft models. Results: TSN-PL nanoparticles of average size 104 nm and encapsulation efficiency approximately 50% showed enhanced dose-dependent cytotoxicity compared with TSN-PTX or clinically used combination of gemcitabine and nano-PTX in two pancreatic cell lines. Significant antitumor efficacy was also established in the pancreatic xenograft model. Conclusion: We developed a unique method of converting total blood serum into chemo drug-loaded nanoparticles and demonstrated its efficacy in vitro and in vivo.

scholarly journals Evaluation of Hsp90 and mTOR inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0248380
Author(s):  
Evelyn M. Mrozek ◽  
Vineeta Bajaj ◽  
Yanan Guo ◽  
Izabela A. Malinowska ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Growth Delay ◽  
Null Cell ◽  
Standard Dosage

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

scholarly journals LMD-07. In Vitro AND In Vivo Culture of Patient Derived-Cerebral Spinal Fluid-Circulating Tumor Cells (PD-CSF-CTCs) in Leptomeningeal Disease (LMD) From Melanoma to Identify Novel Treatment Strategies

2021 ◽  
Vol 3 (Supplement_3) ◽  
pp. iii8-iii8
Author(s):  
Vincent Law ◽  
Zhihua Chen ◽  
Inna Smalley ◽  
Francesca Vena ◽  
Robert Macaulay ◽  
...  
Keyword(s):  
Cell Line ◽  
Treatment Strategies ◽  
Xenograft Model ◽  
Braf Inhibitor ◽  
Mek Inhibitor ◽  
P Value ◽  
Leptomeningeal Disease ◽  
In Vivo Culture

Abstract Background Approximately 5% of melanoma patients (pts) will develop LMD. Currently there is no effective treatments for this disease. A significant barrier to the development of effective therapies has been the inability to culture CSF-CTCs for functional analysis. For the first time, we were able to successfully expand CSF-CTCs in vitro and in vivo. We assessed gene signatures of PD-CSF-CTCs to determine novel targets for therapy. As a proof of concept, we tested the efficacy of combining ceritinib (cer), an IGF-1R inhibitor and trametinib (tra), a MEK inhibitor, against LMD. Methods CSF from 11 pts were collected from various sources (ie: LPs, Ommayas, rapid autopsies). PD-CSF-CTCs were expanded in vitro in conditioned media and in vivo using cell line-derived xenograft model. Single-cell RNA-sequencing (scRNAseq) analysis was performed to assess transcriptional profiles of PD-CSF-CTCs. Results Of the total 61 PD-CSF-CTCs collected from 11 pts (avg: 4.07 CSF collections/patient), we successfully cultured PD-CSF-CTCs from 3 pts (20%) and were able to grow them in vivo from 2 pts (18%). scRNAseq identified IGF-1R, Sox9, ErbB3 and MLANA were among the enriched genes for PD-CSF-CTCs. IGF-1R inhibition by cer and depletion by CRISPR suppressed cell growth. We evaluated the responses of cer + tra treatment in vitro and found that combining these agents produced drug synergy against PD-CSF-CTCs and resensitized BRAF inhibitor-resistant melanoma cell line, WM164R. In vivo LMD xenograft model showed cer + tra treatment significantly prolonged median survival of PD-CSF-CTCs LMD (control: 27 days vs treatment: 38.5 days; P value &lt; 0.032) and WM164R LMD (control: 35 days vs treatment: MS not reached; P value &lt; 0.047). Conclusions Though the sample size is small, this is the first report of the successful in vitro and in vivo culture of CSF-CTCs from pts with LMD.

scholarly journals The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

1999 ◽  
Vol 19 (9) ◽  
pp. 6003-6011 ◽  
Author(s):  
Hélène Talarmin ◽  
Claude Rescan ◽  
Sandrine Cariou ◽  
Denise Glaise ◽  
Giuliana Zanninelli ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Partial Hepatectomy ◽  
Mek Inhibitor ◽  
Signalling Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Mrna Accumulation ◽  
Erk Activation ◽  
Mitogen Activated Protein

ABSTRACT In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G1 phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G1 (30 min to 4 h), and the other occurred in mid-late G1, peaking at around 10.5 h. Interestingly, the mid-late G1 activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G1/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G1 MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G1 MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G1 phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.

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