Increased Saturation Density Research Articles

Space charge saturation in multipactor discharges with parallel magnetic field

Numerical and theoretical models were developed to investigate space-charge effects in a multipactor discharge with an applied magnetic field. The magnetic field was assumed to be parallel to the RF electric field, and only the space-charge effects in the direction perpendicular to the fields were studied. Inclusion of space-charge effects allowed investigation of a saturation mechanism driven by electron diffusion. Numerical simulations showed that saturation of the multipactor discharge could be reached as diffusion balanced the electron production due to secondary emission. It was found that the steady state density reaches a limit as RF voltage was increased. Fluid equations for the electron motion were used to describe electron diffusion and derive an expression for the steady-state electron density in a typical multipactor discharge. It was found that in the presence of a magnetic field, electrons may be trapped by executing a slow rigid-rotor rotation, as described by the Brillouin condition. The electron behavior was found to be dynamic, satisfying the electron trapping condition while the electrons were moving between electrodes and violating the Brillouin condition near the electrodes leading to larger diffusion losses. Numerical simulations show that Brillouin trapping due to an applied parallel magnetic field can significantly increase saturation density by up to two orders of magnitude.

Anti-proliferative and pro-apoptotic effects of rosemary and constituent terpenoids in a model for the HER-2-enriched molecular subtype of clinical breast cancer.

Neoadjuvant treatment options for human epidermal growth factor receptor-2 (HER-2)-enriched and luminal B molecular subtypes of clinical breast cancer include HER-2-targeted therapy with chemotherapy or anti-hormonal therapy. These treatment options result in systemic toxicity and acquired tumor resistance. Minimally toxic naturally occurring phytochemicals may represent testable alternatives to conventional therapy. HER-2-overexpressing tumorigenic human mammary epithelial 184-B5/HER cells represent a model for the HER-2-enriched breast cancer subtype. Non-fractionated rosemary extract (RME) and constituent phenolic terpenoids ursolic acid (UA), carnosol (CSOL) and carnosic acid (CA) represented the test agents. Anchorage-independent (AI) proliferation, cell cycle progression, cellular apoptosis and expression of cell cycle-regulatory and apoptosis-specific proteins represented the mechanistic end point biomarkers. Relative to the parental non-tumorigenic 184-B5 cells, tumorigenic 184-B5/HER cells exhibited decreased population doubling, increased saturation density, accelerated cell cycle progression and downregulated cellular apoptosis, confirming the loss of homeostatic control of proliferation. Treatment with the test agents resulted in a dose-dependent decrease in AI colony number, indicating a decrease in cancer risk. Mechanistically, RME and UA inhibited G1-S phase transition resulting in an increased G1:S+G2/M ratio and decreased cyclin D1 expression. The pro-apoptotic effect of RME and UA was indicated by increased sub-G0 (apoptotic) cell population, and relevant reciprocal modulation, as demonstrated by decreased anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased pro-apoptotic Bcl-2-associated X protein expression. In contrast, treatment with CA and CSOL resulted in cytostatic G2/M arrest and an increase in cyclin B1 expression; thus, naturally-occurring rosemary and its constitutive terpenoids re-establish homeostatic control of proliferation and decrease cancer risk via distinct mechanisms. These data validate an experimental approach to prioritize efficacious natural compounds as testable alternatives for conventional chemo-endocrine and HER-2-targeted therapies in HER-2-enriched breast cancer.

Dynamics of cell transformation in culture and its significance for tumor development in animals

NIH 3T3 cells grown in conventional Dulbecco's modification of Eagle's basal medium (DME) produce no transformed foci when grown to confluence in 10% calf serum (CS). A few cultures were transformed by ras oncogenes when transfected with DNA from neoplastic cells, but they failed to do so in 80 to 90% of the transfections. However, when they were grown in a medium [molecular, cellular, and developmental biology 402 (MCDB 402)] optimized for their clonal growth in minimal serum, they produced transformed foci without transfection in 10% CS, but not in 2% CS. The first response to growth in MCDB 402 in 2% CS in successive rounds of contact inhibition was uniform increases in saturation density of the population. This was followed by the appearance of transformed foci. A systematic study was made of the dynamics of neoplastic progression in various concentrations of CS in a single round of confluence at 2 and 3 wk, followed by three sequential rounds of confluence in 2% CS for 2 wk. There was a linear relationship between CS concentration and saturation density in the first-round cultures and continuing differences in subsequent cultures. The hyperplastic field of normal-looking cells surrounding transformed foci became increasingly permissive for transformation with serial culture. The dynamics show that epigenetic selection is the major driving force of neoplastic development. Cells from dense foci produced malignant fibrosarcomas in mice, thereby exhibiting a positive relationship between transformation in culture and the development of tumors.

Abnormal Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Partially Mimicked Development of TSC2 Neurological Abnormalities.

SummaryTuberous sclerosis complex (TSC) is a disease featuring devastating and therapeutically challenging neurological abnormalities. However, there is a lack of specific neural progenitor cell models for TSC. Here, the pathology of TSC was studied using primitive neural stem cells (pNSCs) from a patient presenting a c.1444-2A>C mutation in TSC2. We found that TSC2 pNSCs had higher proliferative activity and increased PAX6 expression compared with those of control pNSCs. Neurons differentiated from TSC2 pNSCs showed enlargement of the soma, perturbed neurite outgrowth, and abnormal connections among cells. TSC2 astrocytes had increased saturation density and higher proliferative activity. Moreover, the activity of the mTOR pathway was enhanced in pNSCs and induced in neurons and astrocytes. Thus, our results suggested that TSC2 heterozygosity caused neurological malformations in pNSCs, indicating that its heterozygosity might be sufficient for the development of neurological abnormalities in patients.

Abstract P5-14-02: Efficacy of tabebuia avellandae extract on a cell culture model for triple negative breast cancer

Abstract Background: The triple negative breast cancer (TNBC), a molecular subtype of clinical breast cancer consisting of epithelial cells that lack estrogen receptor-α (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) expression, is non-responsive to either endocrine therapy or HER-2 targeted therapy. Chemotherapy is frequently associated with long-term systemic toxicity, acquired tumor resistance and resultant compromised treatment efficacy. These aspects emphasize a need to identify efficacious non-toxic agents for secondary prevention/therapy of TNBC. Non-fractionated aqueous extract from the inner bark of the Tabebuia avellandae (TA) tree found in the Amazon rainforest, available as a dietary supplement under the name of Taheebo or Pau d’arco, has documented efficacy in a cell culture model for the Luminal A breast cancer subtype, as well as for several other cancers. Present study examines the inhibitory effects of the TA extract, and identifies possible mechanistic targets for its efficacy in a cell culture model for TNBC. Nutritional Supplement, Experimental Model and Biomarkers: Lyophilized powder of non-fractionated TA extract from Taheebo Japan, Osaka, Japan, provides the source material for the study. The ER-/PR-/HER-2- MDA-MB-231 cell line represents the cell culture model for TNBC. Anchorage dependent growth, cell cycle progression, status of cell cycle regulatory proteins and anchorage independent colony formation, represent the quantitative biomarkers for efficacy. Results: Relative to the non-tumorigenic 184-B5 human mammary epithelial cells, the tumor derived MDA-MB-231 cells exhibited decreased population doubling time, increased saturation density, decreased G1: S+G2/M ratio and increased S+G2/M: Sub G0 ratio, indicating loss of homeostatic growth control. Additionally, unlike 184-B5 cells, MDA-MB-231 cells exhibited increased anchorage independent growth in vitro and tumor development in vivo, indicating enhanced cancer risk. Treatment of MDA-MB-231 cells with TA resulted in a substantial dose dependent cytostatic growth arrest (IC50:1.0%; IC90: 2.5%). Cell cycle analysis of TA treated cells revealed G1 arrest, leading to a progressive dose dependent increase in the G1: S+G2/M ratio. Mechanistically, TA decreased Cyclin D1 expression and attenuated RB phosphorylation, predicting Cyclin D-CDK4-pRB pathway as a molecular target for efficacy. Furthermore, TA effectively inhibited anchorage independent growth in a dose dependent manner. Conclusions: Present data demonstrated pronounced efficacy of TA as a naturally occurring nutritional substance in a cell culture model for TNBC, and validated TA as a promising non-toxic natural agent for secondary prevention/therapy of clinical TNBC. Citation Format: Nitin T Telang, Hareesh B Nair, George YC Wong. Efficacy of tabebuia avellandae extract on a cell culture model for triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-14-02.

The paradox of the contrasting roles of chronic magnesium deficiency in metabolic disorders and field cancerization.

Magnesium (Mg(2+)) deficiency is common in metabolic disorders such as obesity, type 2 diabetes, and insulin resistance. These disorders are also associated with a high incidence of cancer. Mg(2+) is the regulator par excellence of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell. Because Mg(2+) deficiency inhibits energy production it might be expected to inhibit tumor production. However, the high incidence of cancer in metabolic disorders makes that seem unlikely. In order to understand this seeming paradox, it is important to understand the regulatory role of Mg(2+) in normal and neoplastic cells. Free Mg(2+) is the primary regulator of glycolysis and the Krebs cycle. It also acts as a second messenger for growth factors in regulating protein synthesis. Varying Mg(2+) concentrations result in the same set of coordinated responses as varying serum concentrations. Selection by serial rounds of high cell density or reduced serum concentration at low cell density results in progressive stages of field cancerization. Highly transformed cells proliferate in much lower concentrations of Mg(2+) and grow to much higher saturation densities than normal cells. It remains to be seen whether reduction in Mg(2+) in sparse, exponentially proliferating cultures selects for increases in saturation density and transformed foci.

CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APCMin/CD24 double knockout transgenic mice

Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APC(Min/+) mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation-associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by β-catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis.

Involvement of the transcription factor FoxM1 in contact inhibition

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Although it is generally accepted that contact inhibition plays a pivotal role in maintaining tissue homeostasis, the molecular mechanisms of contact inhibition are still not fully understood. FoxM1 is known as a proliferation-associated transcription factor and is upregulated in many cancer types. Vice versa, anti-proliferative signals, such as TGF-β and differentiation signals decrease FoxM1 expression. Here we investigated the role of FoxM1 in contact inhibition in fibroblasts. We show that protein expression of FoxM1 is severely and rapidly downregulated upon contact inhibition, probably by inhibition of ERK activity, which then leads to decreased expression of cyclin A and polo-like kinase 1. Vice versa, ectopic expression of FoxM1 prevents the decrease in cyclin A and polo-like kinase 1 and causes a two-fold increase in saturation density indicating loss of contact inhibition. Hence, we show that downregulation of FoxM1 is required for contact inhibition by regulating expression of cyclin A and polo-like kinase 1.

Mitochondrial alteration in malignantly transformed human small airway epithelial cells induced by α‐particles

Human small airway epithelial cells (SAECs) immortalized with human telomerase reverse transcriptase were exposed to either a single or multiple doses of α-particles. Irradiated cells showed a dose-dependent cytotoxicity and progressive neoplastic transformation phenotype. These included an increase in saturation density of growth, a greater resistance to N-phosphonoacetyl-L-aspartate, faster anchorage-independent growth, reinforced cell invasion and c-Myc expression. In addition, the transformed cells formed progressively growing tumors upon inoculation into athymic nude mice. Specifically, α-irradiation induced damage to both mitochondrial DNA (mtDNA) and mitochondrial functions in transformed cells as evidenced by increased mtDNA copy number and common deletion, decreased oxidative phosphorylation activity as measured by cytochrome C oxidase (COX) activity and oxygen consumption. There was a linear correlation between mtDNA copy number, common deletion, COX activity and cellular transformation represented by soft agar colony formation and c-Myc expression. These results suggest that mitochondria are associated with neoplastic transformation of SAEC cells induced by α-particles, and that the oncogenesis process may depend not only on the genomes inside the nucleus, but also on the mitochondrial DNA outside the nucleus.

Differentiation-Promoting Culture of Competent and Noncompetent Keratinocytes Identifies Biomarkers for Head and Neck Cancer

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

Fields and field cancerization: The preneoplastic origins of cancer

Most basic research on cancer concerns genetic changes in benign and malignant tumors. Yet evidence indicates that the majority of the mutations in tumors occur in the preneoplastic field stage of their development. That early stage is represented by grossly invisible, broad regions of "field cancerization" which have not, heretofore, been operationally analyzed in cell culture. Conditions are described for quantitating preneoplasia by increased saturation density followed by progression to transformation. These parameters are driven by Darwinian selection of spontaneously occurring, cumulative mutations, in accordance with recent genomic analyses of human cancer, just as it is in the evolution of species. The cell culture model will allow correlation of the preneoplastic increases in saturation density with genetic changes, and development of methods for demarcating fields during surgery so that they can be excised along with the tumor, thereby reducing the possibility of recurrence at the site.

Mutant fibroblast growth factor receptor 3 induces intracellular signaling and cellular transformation in a cell type- and mutation-specific manner

Although activating mutations of FGFR3 are frequent in bladder tumors, little information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum. We investigated the phenotypic and signaling consequences of three FGFR3 mutations (S249C, Y375C, and K652E) in immortalized normal human urothelial cells (TERT-NHUC) and mouse fibroblasts (NIH-3T3). In TERT-NHUC, all mutant forms of FGFR3 induced phosphorylation of FRS2α and ERK1/2, but not AKT or SRC. PLCγ1 phosphorylation was only observed in TERT-NHUC expressing the common S249C and Y375C mutations, and not the rare K652E mutation. Cells expressing S249C and Y375C FGFR3 displayed an increased saturation density, related to increased proliferation and viability. This effect was significantly dependent on PLCγ1 signaling and undetectable in cells expressing K652E FGFR3, which failed to phosphorylate PLCγ1. In contrast to TERT-NHUC, expression of mutant FGFR3 in NIH-3T3 resulted in phosphorylation of Src and Akt. Additionally, all forms of mutant FGFR3 were able to phosphorylate Plcγ1 and induce morphological transformation, cell proliferation, and anchorage independent growth. Our results indicate that the effects of mutant FGFR3 are both cell type- and mutation-specific. Mutant FGFR3 may confer a selective advantage in the urothelium by overcoming normal contact inhibition of proliferation.

Saturation Density of Skin Fibroblasts as a Quantitative Screen for Human Cancer Susceptibility

Genomic analysis of human cancers reveals a large number of genetic changes per cell that presumably underlie development of the disease. The complexity of these changes that differ from one type of cancer to the other and from patient to patient with the same type of cancer raises questions about the feasibility of genomic analysis as an indicator of susceptibility to cancer. However, skin fibroblasts (SF) from individuals in families with heritable forms of cancer, and from cancer-bearing individuals, show correlation with a significant increase in saturation density (SD), as well as other neoplasia-related properties. Procedures are described for amplifying and quantifying differences in SD on the basis of studies of spontaneous transformation in the NIH 3T3 line of mouse fibroblasts. It is proposed that such procedures be evaluated as quantitative screens for susceptibility to cancer in the general population.

Genes Involved in Differentiation, Stem Cell Renewal, and Tumorigenesis Are Modulated in Telomerase-Immortalized Human Urothelial Cells

The expression of hTERT, the catalytic subunit of telomerase, immortalizes normal human urothelial cells (NHUC). Expression of a modified hTERT, without the ability to act in telomere maintenance, did not immortalize NHUC, confirming that effects at telomeres are required for urothelial immortalization. Previous studies indicate that inhibition of telomerase has an immediate effect on urothelial carcinoma (UC) cell line viability, before sufficient divisions to account for telomere attrition, implicating non-telomere effects of telomerase in UC. We analyzed the effects of telomerase on gene expression in isogenic mortal and hTERT-transduced NHUC. hTERT expression led to consistent alterations in the expression of genes predicted to be of phenotypic significance in tumorigenesis. A subset of expression changes were detected soon after transduction with hTERT and persisted with continued culture. These genes (NME5, PSCA, TSPYL5, LY75, IGFBP2, IGF2, CEACAM6, XG, NOX5, KAL1, and HPGD) include eight previously identified as polycomb group targets. TERT-NHUC showed overexpression of the polycomb repressor complex (PRC1 and PRC4) components, BMI1 and SIRT1, and down-regulation of multiple PRC targets and genes associated with differentiation. TERT-NHUC at 100 population doublings, but not soon after transduction, showed increased saturation density and an attenuated differentiation response, indicating that these are not acute effects of telomerase expression. Some of the changes in gene expression identified may contribute to tumorigenesis. Expression of NME5 and NDN was down-regulated in UC cell lines and tumors. Our data supports the concept of both telomere-based and non-telomere effects of telomerase and provides further rationale for the use of telomerase inhibitors in UC.

Combinatorial prevention of carcinogenic risk in a model for familial colon cancer

Germ line mutations in the tumor suppressor adenomatous polyposis coli (APC) gene, predispose for the clinical familial adenomatous polyposis (FAP) syndrome, a high risk precursor for early onset colon cancer. Similar mutations in the murine homolog of the APC gene, however, produce adenomas predominantly in the small intestine, rather than in the colon. The objectives of the present study were: i) to develop a preclinical cell culture model for human FAP syndrome and ii) to validate this model as a rapid mechanism-based approach for evaluation of the preventive efficacy of combinations of synthetic pharmacological agents or naturally-occurring phytochemicals, for the risk of colon carcinogenesis. The clonally selected 850Min COL-Cl1 cell line derived from histologically normal colon of ApcMin/+ mouse exhibited aberrant proliferation (64.7% decrease in population doubling time, 820% increase in saturation density, and 81.4% decrease in spontaneous apoptosis), relative to that observed in the colon epithelial cell line C57 COL established from Apc [+/+] C57BL/6J mouse. In addition, unlike the Apc [+/+] C57 COL cells, the Apc mutant cells exhibited enhanced risk for spontaneous carcinogenic transformation as evidenced by 100% increase in anchorage-independent colony formation (C57 COL: 0/12; 850Min COL-Cl1: 12/12, mean colony number 23.6+/-2.7). Treatment of Apc mutant cells with low dose combination of select mechanistically distinct synthetic chemopreventive agents such as celecoxib (CLX) + difluoro methylornithine (DFMO), or naturally-occurring epigallocatechin gallate (EGCG) + curcumin (CUR) produced 160-400% and 220-430% decrease in the viable cell number respectively, relative to these agents used independently. Furthermore, relative to independent agents, CLX+DFMO and EGCG+CUR combinations produced 31.5-82.1% and 45.9-105.4% greater reduction in the number of anchorage-independent colonies. Thus, aberrant proliferation and increased risk for carcinogenesis in the Apc mutant cells, and their susceptibility to low dose combinations of mechanistically distinct chemopreventive agents validate a rapid approach to prioritize efficacious combinations for long-term animal studies and future clinical trials on prevention of colon cancer.

Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism

The antizyme inhibitor was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Blocking antizyme activity subsequently leads to increased intracellular levels of ODC and increased ODC enzymatic activity. We now report that antizyme inhibitor is a positive modulator of cell growth. Overexpression of antizyme inhibitor in NIH-3T3 mouse fibroblasts or in AT2.1 Dunning rat prostate carcinoma cells resulted in an increased rate of cell proliferation and an increase in saturation density of the cultured cells. This was accompanied by an increase in intracellular levels of the polyamine putrescine. In AT2.1 cells, antizyme inhibitor overexpression also increased the ability of the cells to form foci when grown under anchorage-independent conditions. In order to determine the role of antizyme on antizyme inhibitor activity we created an antizyme inhibitor mutant, AZI(Delta117-140), which lacks the putative antizyme-binding domain. We show that this mutant fails to bind to antizyme, but remains capable of inducing increased rates of cell proliferation, suggesting that antizyme inhibitor has antizyme-independent functions. Silencing antizyme inhibitor expression leads to diminished levels of cyclin D1 and to reduced cell proliferation. Antizyme inhibitor is capable of preventing cyclin D1 degradation, and this effect is at least partially independent of antizyme. We show that wild-type antizyme inhibitor and the AZI(DeltaY) mutant are capable of direct interaction with cyclin D1 suggesting a potential mechanism for the antizyme-independent effects. Together, our data suggest a novel function for antizyme inhibitor in cellular growth control.

Multiple Oncogenic Changes (K-RASV12, p53 Knockdown,Mutant EGFRs, p16Bypass, Telomerase) Are Not Sufficient to Confer a Full Malignant Phenotype on Human Bronchial Epithelial Cells

We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention.

Expression of the Smad3 Transgene Restores Radiosensitivity and Migratory Capacity to a Smad3−/− Clonal Bone Marrow Stromal Cell Line.

An intact Smad3 gene product is critical for a functioning signal transduction pathway following TGF binding to the TGF-β receptor. We have previously established Smad3−/− and Smad3+/+ long term bone marrow cultures (LTBMCs) and isolated clonal bone marrow stromal cell lines from each. The Smad3−/− cells were smaller in size but had a faster cell doubling time (24 hours compared to 48 hours) and increased saturation density compared to +/+ cells (15.3 ± 1.0 x 105 cells/25 mm2 flask compared to 3.8 ± 0.1 x 105, p = 0.003). The plating efficiency of the lines was similar (18.3 ± 2.7 compared to 15.5 ± 1.7, p = 0.417). We transfected the Smad3−/− cell line with a retrovirus containing the Smad3 transgene, and selected a subclone expressing the transgene mRNA, designated Smad3−/−(3). Smad3−/−(3) cells were increased in size to that of Smad3+/+ cells, and showed decreased cell saturation density. Using the Cytoworks computer controlled cell tracking Bioreactor, we measured the migration of each clonal line. Tissue culture wells of 100 cells per well were followed for 5 days tracking each cell in quadruplicate wells per cell line. Smad3+/+ cells migrated significantly faster over 5 days in culture compared to Smad3−/− cells. (The average velocities were 0.62 μm/min for Smad3+/+ and 0.36 μm/min for Smad3−/−, p<0.0001). Over 5 days, the average velocities for Smad3+/+ cells were 0.51, 0.51, 0.52, 0.72, 0.91 μm/min, and for Smad3−/− cells were 0.28, 0.38, 0.41, 0.37, 0.35 μm/min. The 5 p-values comparing these cell lines were all <0.0001. The 7 day clonagenic irradiation survival curve showed that Smad3+/+ and Smad3−/−(3) cells were significantly more sensitive (D0 = 1.75 ± 0.03 and 1.51 ± 0.07 Gy, respectively) compared to the Smad3−/− cell line (D0 = 2.43 ± 0.06 Gy, p=0.0016 and 0.0103). These results demonstrate a concordance of radioresistance and decreased migratory capacity in bone marrow stromal cells devoid of a functioning Smad3 gene product, and restoration of both properties following overexpression of the transgene product. These data may help explain the decreased radiation fibrosis observed in Smad3−/− mice.

Degrees and kinds of selection in spontaneous neoplastic transformation: an operational analysis.

Spontaneous neoplastic transformation develops within days in the NIH 3T3 line of cells through differential inhibition of their proliferation under contact inhibition. A small fraction of the population continues to multiply after saturation density is reached and is selected to progressively increase saturation density in successive rounds of confluence. The degree of selection at confluence depends on the extent of proliferation of some cells in a heterogeneous population. The development of transformed foci is an extension of the same selective process that increases saturation density. The expression of the foci is enhanced with increases in the saturation density of the surrounding cells. Transformation is also induced by moderately reducing the concentration of calf serum in the medium during low-density passages, which allows selection of cells that require less growth factor. Further stepwise reductions in serum increase the degree of transformation. Contact inhibition and reduction in serum concentration select for the same phenotype of cell that increases saturation density and generates transformed foci. There is mounting evidence that selection is a major factor in the development of common epithelial tumors of humans, but it extends over decades rather than days, and the in vivo microenvironment selects from more stable populations of cells than those in culture. The many progressive levels of increased saturation density and transformed focus formation suggest that a very large number of genes participate in neoplastic development. The operational model of variation and selection presented here may aid in understanding chemical carcinogenesis and cancer recurrence after chemotherapy.

High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27 Kip Cdk-complexes

The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27 Kip1 levels rose and the inhibitor accumulated in both Cdk4 and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27 Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density.