Search

Germline-activating mutations in HRAS cause Costello syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here, we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via reactive oxygen species-dependent AMP-activated protein kinase α and p38 hyperactivation, occurs in primary fibroblasts of CS patients, resulting in accelerated glycolysis and increased fatty acid synthesis and storage as lipid droplets. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.

Terminal differentiation is an ill-defined, insufficiently characterized, nonproliferation state. Although it has been classically deemed irreversible, it is now clear that at least several terminally differentiated (TD) cell types can be brought back into the cell cycle. We are striving to uncover the molecular bases of terminal differentiation, whose fundamental understanding is a goal in itself. In addition, the field has sought to acquire the ability to make TD cells proliferate. Attaining this end would probe the very molecular mechanisms we are trying to understand. Equally important, it would be invaluable in regenerative medicine, for tissues depending on TD cells and devoid of significant self-repair capabilities. The skeletal muscle has long been used as a model system to investigate the molecular foundations of terminal differentiation. Here, we summarize more than 50 years of studies in this field.

Terminally differentiated cells are defined by their inability to proliferate. When forced to re-enter the cell cycle, they generally cannot undergo long-term replication. Our previous work with myotubes has shown that these cells fail to proliferate because of their intrinsic inability to complete DNA replication. Moreover, we have reported pronounced modifications of deoxynucleotide metabolism during myogenesis. Here we investigate the causes of incomplete DNA duplication in cell cycle-reactivated myotubes (rMt). We find that rMt possess extremely low levels of thymidine triphosphate (dTTP), resulting in very slow replication fork rates. Exogenous administration of thymidine or forced expression of thymidine kinase increases deoxynucleotide availability, allowing extended and faster DNA replication. Inadequate dTTP levels are caused by selective, differentiation-dependent, cell cycle-resistant suppression of genes encoding critical synthetic enzymes, chief among which is thymidine kinase 1. We conclude that lack of dTTP is at least partially responsible for the inability of myotubes to proliferate and speculate that it constitutes an emergency barrier against unwarranted DNA replication in terminally differentiated cells.

SummaryHepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.

Tissue repair and regeneration are very complex biological events, whose successful attainment requires far more than mere cell division. However, almost unavoidably they entail cell proliferation as a fundamental premise. Full regeneration or repair cannot be achieved without replacing cells lost to disease or injury, replacement that can only take place via proliferation of surviving cells. This review endeavors to outline the molecular bases of exit from and reentry into the cell cycle. In recent years, the decision to proliferate or not has been seen as mostly the concern of cyclins and cyclin-dependent kinases. This account tries to show that cell cycle inhibitors are as important as the positive regulators in the making of this decision. Finally, the authors wish to suggest that the molecular knowledge of the cell cycle can be harnessed to the benefit of many aspects of regenerative medicine. (Part of Multi-author Review)

The differentiation of skeletal myoblasts is characterized by permanent withdrawal from the cell cycle and fusion into multinucleated myotubes. Muscle cell survival is critically dependent on the ability of cells to respond to oxidative stress. Base excision repair (BER) is the main repair mechanism of oxidative DNA damage. In this study, we compared the levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8-hydroxyguanine DNA levels in terminally differentiated cells, suggested that reactive oxygen species are produced during this process. The repair of 2-deoxyribonolactone, which is exclusively processed by long-patch BER, was impaired in cell extracts from myotubes. The repair of a natural abasic site (a preferred substrate for short-patch BER) also was delayed. The defect in BER of terminally differentiated muscle cells was ascribed to the nearly complete lack of DNA ligase I and to the strong down-regulation of XRCC1 with subsequent destabilization of DNA ligase IIIα. The attenuation of BER in myotubes was associated with significant accumulation of DNA damage as detected by increased DNA single-strand breaks and phosphorylated H2AX nuclear foci upon exposure to hydrogen peroxide. We propose that in skeletal muscle exacerbated by free radical injury, the accumulation of DNA repair intermediates, due to attenuated BER, might contribute to myofiber degeneration as seen in sarcopenia and many muscle disorders.

In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference–mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D–cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle–driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.

Although in the last decades the molecular underpinnings of the cell cycle have been unraveled, the acquired knowledge has been rarely translated into practical applications. Here, we investigate the feasibility and safety of triggering proliferation in vivo by temporary suppression of the cyclin-dependent kinase inhibitor, p21. Adeno-associated virus (AAV)-mediated, acute knockdown of p21 in intact skeletal muscles elicited proliferation of multiple, otherwise quiescent cell types, notably including satellite cells. Compared with controls, p21-suppressed muscles exhibited a striking two- to threefold expansion in cellularity and increased fiber numbers by 10 days post-transduction, with no detectable inflammation. These changes partially persisted for at least 60 days, indicating that the muscles had undergone lasting modifications. Furthermore, morphological hyperplasia was accompanied by 20% increases in maximum strength and resistance to fatigue. To assess the safety of transiently suppressing p21, cells subjected to p21 knockdown in vitro were analyzed for γ-H2AX accumulation, DNA fragmentation, cytogenetic abnormalities, ploidy, and mutations. Moreover, the differentiation competence of p21-suppressed myoblasts was investigated. These assays confirmed that transient suppression of p21 causes no genetic damage and does not impair differentiation. Our results establish the basis for further exploring the manipulation of the cell cycle as a strategy in regenerative medicine.

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.

Background Terminally differentiated (TD) cells permanently exit the mitotic cycle while acquiring specialized characteristics. Although TD cells can be forced to reenter the cell cycle by different means, they cannot be made to stably proliferate, as attempts to induce their replication constantly result in cell death or indefinite growth arrest. There is currently no biological explanation for this failure. Principal Findings Here we show that TD mouse myotubes, reactivated by depletion of the p21 and p27 cell cycle inhibitors, are unable to complete DNA replication and sustain heavy DNA damage, which triggers apoptosis or results in mitotic catastrophe. In striking contrast, quiescent, non-TD fibroblasts and myoblasts, reactivated in the same way, fully replicate their DNA, do not suffer DNA damage, and proliferate even in the absence of growth factors. Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4. Conclusions We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method. On reviewing the literature concerning reactivation of other TD cell types, we propose that similar mechanisms underlie the general inability of all kinds of TD cells to proliferate in response to otherwise mitogenic stimuli. These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate. Furthermore, this trait might contribute to explain the inability of these cells to play a role in tissue repair, unlike their counterparts in extensively regenerating species.

The molecular anatomy of cancer cells is being explored through unbiased approaches aimed at the identification of cancer-specific transcriptional signatures. An alternative biased approach is exploitation of molecular tools capable of inducing cellular transformation. Transcriptional signatures thus identified can be readily validated in real cancers and more easily reverse-engineered into signaling pathways, given preexisting molecular knowledge. We exploited the ability of the adenovirus early region 1 A protein (E1A) oncogene to force the reentry into the cell cycle of terminally differentiated cells in order to identify and characterize genes whose expression is upregulated in this process. A subset of these genes was activated through a retinoblastoma protein/E2 viral promoter required factor–independent (pRb/E2F-independent) mechanism and was overexpressed in a fraction of human cancers. Furthermore, this overexpression correlated with tumor progression in colon cancer, and 2 of these genes predicted unfavorable prognosis in breast cancer. A proof of principle biological validation was performed on one of the genes of the signature, skeletal muscle cell reentry-induced (SKIN) gene, a previously undescribed gene. SKIN was found overexpressed in some primary tumors and tumor cell lines and was amplified in a fraction of colon adenocarcinomas. Furthermore, knockdown of SKIN caused selective growth suppression in overexpressing tumor cell lines but not in tumor lines expressing physiological levels of the transcript. Thus, SKIN is a candidate oncogene in human cancer.

Cyclin E is a key cell cycle regulator, whose accumulation is believed to be positively modulated chiefly at the transcriptional level. We show that forced expression of E2F family members specifically stabilizes cyclin E protein by reducing its conjugation with ubiquitin, leading to increased steady-state levels. The stabilized protein shows enhanced association with cdk2 and higher kinase activity, indicating that cyclin E stabilization bears functional consequences. Although the activity of E2F on the cyclin E protein does not entail increased cyclin E gene transcription, it does require the E2F transactivation capacity, as demonstrated by E2F and DP-1 mutant analysis. However, such activity does not require E2F binding to pRb. Furthermore, E2F stabilizes cyclin E even in nonproliferating cells. Our results bear significance for the understanding of tumor progression, in light of the well-known autoregulatory loop between E2F and cyclin E and the disregulation of E2F that is one consequence of the almost universal impairment of the pRb pathway in cancer. Constitutive pRb inactivation leads to enhanced E2F activity through loss of binding. We propose that such increased E2F activity stabilizes cyclin E and contributes to establish the high and persistent levels of the protein commonly found in human neoplasias.

In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism.Acute Rb excision from conditional knockout myotubes caused reexpression of E2F transcriptional activity, cyclin-E and -A kinase activities, PCNA, DNA ligase I, RPA, and MCM2, but did not induce DNA synthesis, showing that pRb is not indispensable to preserve the postmitotic state of these cells. Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program. Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules. Finally, Rb removal induced no DNA synthesis even in pocket-protein null cells. Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects.

Signal Transduction via the hepatocyte growth factor (HGF)/Met receptor kinase pathway is critical for normal embryogenesis and adult homeostasis, and aberrant HGF/Met signaling contributes to tumorigenesis and metastasis in many prevalent forms of cancer. By defining the basic molecular mechanisms of pathway activation at the target cell surface, we hope to develop novel strategies to antagonize oncogenic signaling as well as methods to assess pathway status in cancer patients. These methods may help identify those most likely to benefit from HGF/Met targeted therapies and help monitor treatment response. Distinct domains of HGF bind to the Met receptor tyrosine kinase and to cell-surface heparan sulfate (HS) proteoglycans. The latter are widely expressed, structurally diverse biopolymers that modulate many important protein-protein interactions, but their importance in eliciting HGF biological responses is controversial. We have identified basic (positively charged) amino acid residues in HGF that form the primary binding site for (negatively charged) HS proteoglycans. HS binding by these residues increases the affinity of ligand-receptor binding and, with HS polymers of sufficient length, facilitates ligand oligomerization. Ligand oligomerization, in turn, controls Met kinase activation, cell motility and proliferation under normal conditions. Opposite (negative) charge amino acid substitutions at these critical HS binding residues in HGF can mimic bound HS in promoting high affinity receptor binding, but antagonize the binding of long HS polymers on the cell surface, thereby antagonizing ligand oligomerization and its consequences. Engineering these substitutions in the context of a short HGF isoform containing only the primary HS and Met binding domains yields a potent and selective competitive inhibitor of oncogenic HGF signaling in vivo. These findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development that may be applicable to other HS-binding growth factors. The Met receptor kinase is among many transmembrane proteins that are proteolytically released from the cell surface by a process known as ectodomain shedding. Consistent with the concept that Met ectodomain shedding is a normal physiological process, we found that healthy human volunteers (n > 100) display soluble Met (sMet) plasma concentrations in the range of 100 - 300 ng/ml and urinary sMet concentrations that are approximately 1000-fold lower. We hypothesized that aberrantly high Met expression, a frequent occurrence in many forms of cancer, and/or dysregulated proteolytic activity, another common feature of aggressive malignancies, could lead to increased ectodomain shedding and that sMet levels could be a useful biomarker of tumorigenesis, tumor progression and overall tumor burden. In prior studies we found that increased Met shedding correlated with malignancy in several cell lines, and that urinary sMet levels were significantly elevated in metastatic prostate cancer patients relative to those with organ-confined cancer or no evidence of disease. In an ongoing investigation of sMet as a diagnostic and/or prognostic biomarker of bladder cancer, the mean urinary sMet level among 150 patients with pathologically confirmed disease was significantly higher than that obtained from a group of 50 individuals with no evidence of disease, whether mean values were normalized to urine creatinine concentration or not (two-tailed Mann-Whitney t-test p-value We have also measured plasma sMet levels in patients participating in a phase II clinical trial of Foretinib (formerly GSK1363089/XL880), a small molecule multikinase inhibitor targeting Met and vascular endothelial cell growth factor-2 (VEGFR-2) Axl and Ron, designed to evaluate the safety and efficacy of 2 dosing schedules (continuous daily dosing and intermittent 5 days on/9 days off dosing) of foretinib as a single therapeutic agent in patients with metastatic gastric carcinoma (GC). Plasma sMet concentrations, as well as plasma VEGF-A, HGF and soluble VEGFR-2 (sVEGFR-2) levels were measured before and during treatment with foretinib to measure potential pharmacodynamic (PD) effects and/or the modulation by foretinib of Met and VEGFR-2 pathways. Marker changes from baseline were analyzed at days 5, 15, 29 and 47 using analysis of variance and their relationships with plasma concentrations of foretinib (PK) and clinical outcome (sum of longest diameter [SLD] of tumor as a surrogate for tumor shrinkage, progression-free survival [PFS], and RECIST) were also examined. In GC patients on the intermittent dosing schedule, plasma sMet and VEGF-A concentrations tended to increase during the treatment periods compared with the drug holidays, suggestive of a direct short-term drug effect on sMet and VEGF-A levels. Although mean tumor burden did not change significantly from baseline to week 8, plasma levels of sMet and VEGF-A correlated positively with the change in SLD over this period, suggesting that the increases of sMet and VEGF-A may be reflective of increased GC tumor burden. Citation Format: Fabiola Cecchi, Deborah Pajalunga, Brian McNeil, Daniel Rabe, Andrew Fowler, Yuan Liu, C. Robert Gagnon, Howard Kallender, Manish A. Shah, Robert Getzenberg, Mark Schoenberg, Anne-Marie Martin, Ramaprasad Srinivasan, W. Marston Linehan, R Andrew Byrd, Donald P. Bottaro. Oncogenic signal transduction via the hepatocyte growth factor/Met receptor kinase pathway [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY23-03

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth. We now show that HGF signaling requires specific HS binding interactions and that targeted disruption of these HS binding sites in HGF yields a potent and selective competitive inhibitor of normal and oncogenic HGF signaling. Our findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development which may be applicable to other HS-binding growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 342.

Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth, and inhibition of these activities by 3M NK1 validates a novel strategy for developing antagonists of the HGF pathway and potentially those of other HS-binding ligands Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B228.

We have recently shown that all kinds of non-proliferating cells, including quiescent, senescent, and terminally differentiated ones, can be mitotically reactivated by the sole removal of cell type-specific cyclin-dependent kinase inhibitors. Reactivation takes place irrespective of added growth factors, allowing otherwise quiescent or senescent cells to proliferate. These unexpected findings warrant a reappraisal of some key aspects of the cell cycle. Inhibitors do not only modulate kinase activity, but contribute to the decision to enter the cell cycle as much as cyclins themselves. Non-proliferating cells, even those destined never to reenter the cell cycle, continue to express functionally significant levels of pre-assembled cyclin-cdk complexes, making cell cycle-arrest a state that must be constantly maintained by active expression of cyclin-dependent kinase inhibitors (CKIs). In addition, we suggest that the novel findings can be exploited in human therapy to accelerate, promote, or induce cell proliferation, both in vitro and in vivo. They should prove advantageous in cell biotechnology, cell replacement therapy, and tissue repair, wherever cell proliferation constitutes a limiting factor.