Reduction In Colony Forming Efficiency Research Articles

Abstract A22: Differential MYC dependence in NSCLC identified through pharmacological and genetic MYC inhibition

Abstract MYC is one of the most commonly deregulated oncogenes in various cancers, including breast, colorectal and lung. While mutations are rare, we know that MYC is overexpressed and in some cases amplified in these (and other) cancers. Numerous reports have recently demonstrated the utility of various therapeutics in selectively targeting MYC-driven cancers. However, given the lack of consistency across tissue types, particularly lung cancer, a multimodal approach to delineate MYC-dependent lung cancers is required. Our goal is to identify lung cancers that are addicted to MYC, determine their oncogenotype and molecular properties, and identify a biomarker to identify patients with MYC addicted tumors. We studied a large panel of clinically and molecularly annotated NSCLC lines for MYC mRNA, protein expression, and DNA copy number. Functional tests were performed on 17 NSCLC cell lines using three drugs that were recently shown to selectively target MYC-driven cancers. Further, we utilized the dominant negative mini-protein OMOMYC for functional classification. In all cases, effects were monitored by colony forming efficiency (CFE) assays and proliferation assays. OMOMYC results were confirmed via xenograft experiments. Each of the three MYC inhibitors tested elicited a viability response in a subset of the 17 NSCLC cell lines, though the sensitive subset was not significantly similar between any two drugs (highest correlation coefficient of 0.3). In order to determine which, if any, of the drugs truly targeted MYC-driven lung cancers, we stably expressed OMOMYC in all 17 parental NSCLC cells and performed functional assays. 6/17 cell lines were dramatically sensitive to OMOMYC (with up to 100 fold reduction in CFE), compared to 11/17 totally resistant. The viability in the presence of OMOMYC shows a statistically significant correlation with one of the three MYC inhibitors tested, which supports the notion that this sensitive subset represents a truly MYC-dependent class of lung cancers. Surprisingly, there was no correlation between MYC dependence and either MYC mRNA, protein expression or DNA copy number. OMOMYC levels were normalized in all cell lines tested and quantified using qRT-PCR. Additionally, in all cases, exogenous OMOMYC expression led to down regulation of c-Myc target genes as measured by both qRT-PCR and microarray. These data suggest that the observed phenotype was the result of decreased MYC activity. We conclude: there is a subset of NSCLCs that demonstrates dramatic growth inhibition by a single MYC-inhibitor, and these data are phenocopied by the more specific MYC-dominant negative protein, OMOMYC. We are classifying this subset of cancer cell lines, MYC “addicted.” Using the MYC “addicted” vs. non- addicted NSCLC panel, we are testing for gene expression, methylation and mutational differences in order to identify and eventually characterize a biomarker for MYC dependence in lung cancer. Citation Format: Patrick Dospoy, Chunli Shao, Elizabeth McMillan, Michael Peyton, Jill Larsen, Luc Girard, Ignacio Wistuba, Adi F. Gazdar, John D. Minna. Differential MYC dependence in NSCLC identified through pharmacological and genetic MYC inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A22.

Dysregulation of the Repressive H3K27 Trimethylation Mark in Head and Neck Squamous Cell Carcinoma Contributes to Dysregulated Squamous Differentiation

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers diagnosed worldwide and is associated with a 5-year survival rate of 55%. EZH2, a component of the polycomb repressor complex 2, trimethylates H3K27 (H3K27me3), which has been shown to drive squamous differentiation in normal keratinocytes. This study determined whether inhibition of EZH2-mediated epigenetic silencing could induce differentiation or provide therapeutic benefit in HNSCC. We determined the effects of inhibiting EZH2, by either RNA interference or pharmacologically, on HNSCC growth, viability, and differentiation in vitro. Xenografts of HNSCC cell lines were used to assess efficacy of 3-deazaneplanocin A (DZNep), an inhibitor of H3K27 trimethylation, in vivo. EZH2 was highly expressed in HNSCC cell lines in vitro and tissue microarray analysis revealed high expression in (n = 59) in situ relative to normal oral epithelium (n = 12). Inhibition of EZH2 with siRNA could induce expression of differentiation genes in differentiation-refractory squamous cell carcinoma cell lines. Differentiation-refractory HNSCC cell lines displayed persistent H3K27me3 on the promoters of differentiation genes. DZNep caused cancer-cell-specific apoptosis in addition to a profound reduction in colony-forming efficiency and induction of some squamous differentiation genes. Furthermore, in vivo, DZNep attenuated tumor growth in two different xenograft models, caused intratumor inhibition of EZH2, and induction of differentiation genes in situ. Collectively, these data suggest that aberrant differentiation in HNSCC may be attributed to epigenetic dysregulation and suggest that inhibition of PRC2-mediated gene repression may represent a potential therapeutic target.

Dependence of Corneal Stem/Progenitor Cells on Ocular Surface Innervation

Neurotrophic keratopathy (NK) is a corneal degeneration associated with corneal nerve dysfunction. It can cause corneal epithelial defects, stromal thinning, and perforation. However, it is not clear if and to which extent epithelial stem cells are affected in NK. The purpose of this study was to identify the relationship between corneolimbal epithelial progenitor/stem cells and sensory nerves using a denervated mouse model of NK. NK was induced in mice by electrocoagulation of the ophthalmic branch of the trigeminal nerve. The absence of corneal nerves was confirmed with β-III tubulin immunostaining and blink reflex test after 7 days. ATP-binding cassette subfamily G member 2 (ABCG2), p63, and hairy enhancer of split 1 (Hes1) were chosen as corneolimbal stem/progenitor cell markers and assessed in denervated mice versus controls by immunofluorescent microscopy and real-time PCR. In addition, corneolimbal stem/progenitor cells were detected as side population cells using flow cytometry, and colony-forming efficiency assay was performed to assess their function. ABCG2, p63, and Hes1 immunostaining were significantly decreased in denervated eyes after 7 days. Similarly, the expression levels of ABCG2, p63, K15, Hes1, and N-cadherin transcripts were also significantly decreased in denervated eyes. Stem/progenitor cells measured as side population from NK mice were decreased by approximately 75% compared with normals. In addition, the authors found a significant (P = 0.038) reduction in colony-forming efficiency of stem/progenitor cells harvested from denervated eyes. Corneolimbal stem/progenitor cells are significantly reduced after depletion of sensory nerves. The data suggest a critical role of innervation in maintaining stem cells and/or the stem cell niche.

The nuclear to cytoplasmic shift of ING5 protein during colorectal carcinogenesis with their distinct links to pathologic behaviors of carcinomas

Inhibitor of growth 5, a tumor suppressor protein, can interact with p53, thereby inhibiting cell growth and inducing apoptosis. Inhibitor of growth 5 overexpression results in a reduction in colony-forming efficiency and cell population in S phase. To clarify the roles of inhibitor of growth 5 in tumorigenesis and progression of colorectal carcinomas, we examined inhibitor of growth 5 expression by immunohistochemistry on a tissue microarray containing colorectal carcinomas (n = 306), adenomas (n = 69), and nonneoplastic mucosa (n = 288) and compared this with clinicopathologic parameters of the carcinomas. In addition, inhibitor of growth 5 expression in colorectal carcinoma tissues and cell lines (DLD-1, HCT-15, SW480, and WiDr) was analyzed by Western blot and reverse transcriptase-polymerase chain reaction. It was found that the inhibitor of growth 5 protein was localized to the nuclei of colon carcinoma cells with no differences at mRNA levels. Among 18 frozen samples of colorectal carcinoma, significantly increased expression of inhibitor of growth 5 protein was observed in the carcinoma in comparison with adjacent mucosa in 14 cases (77.8%; P < .05), and 71.4% (10/14) of carcinoma cases exhibited up-regulated inhibitor of growth 5 mRNA expression. Decreased inhibitor of growth 5 expression was detected by immunohistochemistry in colorectal carcinoma, compared with non-neoplastic mucosa and adenoma (P < .05). Nuclear inhibitor of growth 5 expression was negatively correlated with tumor size, depth of invasion, degree of dedifferentiation, and Union Internationale Contre le Cancer staging (P < .05). In contrast, cytoplasmic inhibitor of growth 5 expression was positively correlated with depth of invasion, lymphatic invasion, and Union Internationale Contre le Cancer staging (P < .05). It was suggested that aberrant inhibitor of growth 5 expression may contribute to pathogenesis, growth, and invasion of colorectal carcinomas and could be considered as a promising marker to gauge aggressiveness of colorectal carcinomas.

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01–25 μg/ml) caused from 15% to > 100% increases in CFE of RTE cells. At high concentrations (100–200 μg/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50–200 μg/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4 5 (50 μg/ml) and 6 8 (100 μg/ml) experiments. The in vivo exposure of rats to NNK (150–450 mg/kg, given i.p.) resulted in a 60–85% reduction in CFE and a 3–5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3 3 and 2 3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.

Comparison of response of bovine and porcine pulmonary arterial endothelial cells to monocrotaline pyrrole

Monocrotaline (MCT)-induced vascular injury in liver and lung may be caused by interaction of MCT metabolites such as monocrotaline pyrrole (MCTP) with vascular cells. Responses of bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively) to a single administration of MCTP were compared. MCTP caused a delayed and progressive release of lactate dehydrogenase (LDH) activity from BECs and a gradual decrease in monolayer cellularity. Surviving cells became markedly hypertrophic. PECs were less sensitive to the cytolytic effects of MCTP, showing minimal cell detachment and little release of LDH activity. However, monolayer cellularity, as assessed by PEC enumeration, decreased in a dose-dependent manner. Hypertrophy of surviving PECs was less pronounced than in BECs. MCTP caused enhanced release of prostacyclin from monolayers of BECs and PECs exposed to 10 micrograms MCTP/ml, and concentrations of 0.5 microgram/ml or greater caused equivalent reduction in colony-forming efficiency in both cell types. In summary, whereas BECs were more susceptible to the cytolytic and hypertrophic effects of MCTP, BECs and PECs responded similarly with regard to prostacyclin release and were equally sensitive to the cytostatic effects of this compound.

遺伝子組み賛え Tumor Necrosis Factor α(rTNFα) のヒト腎細胞癌株KU-2に対する細胞障害性の検討

We attempted to clarify the anti-tumor activity and its mechanism of human recombinant tumor necrosis factor alpha (rTNF alpha). The established cell line KU-2, derived from human renal cell carcinoma, was treated with rTNF alpha alone or in combination with the following anti-cancer agents in vitro: actinomycin D (ACD), vinblastine sulfate (VLB), nimustine hydrochloride (ACNU), and methotrexate (MTX). In vitro studies, including cytotoxic assay, colony forming assay and flow cytometric DNA analysis were performed. By cytotoxic assay, 21.4 +/- 4.0% and 34.8 +/- 4.7% of the cells were killed by 72 hour incubation with 100 ng/ml of rTNF alpha alone, and 1 ng/ml of ACD alone, respectively. An augmented cytotoxicity of 75.3 +/- 0.3% was observed by simultaneously adding 1 ng/ml of rTNF alpha and 1 ng/ml ACD. However, when KU-2 was treated with both 100 ng/ml of rTNF alpha and 3 micrograms/ml of ACNU or 2.5 ng/ml of MTX, no significant increase in cytotoxicity was noted. In the colony forming assay study, the colony forming efficiency (CE) of the control cultures was 31.8 +/- 8.1%. A 92.3 +/- 1.8% reduction in CE was demonstrated when 100 ng/ml of rTNF alpha was added to the cultures. No augmented effects were seen between rTNF alpha and chemotherapeutic agents in this study. In flow cytometric DNA analysis, no cell cycle specific effects of rTNF alpha were demonstrated, regardless of whether or not chemotherapeutic agents were added. These results indicate that the cytotoxic and cytostatic activities of rTNF alpha may be mediated by separate mechanisms of action and that rTNF alpha affects more markedly KU-2 cells having clonogenic potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth‐inhibiting effect of tumor necrosis factor on human umbilical vein endothelial cells is enhanced with advancing age in vitro

We have examined the effects of in vitro aging on the growth capacity of human umbilical vein endothelial cells (HUVECs) under the influence of tumor necrosis factor (TNF) with or without interferon-gamma (IFN-gamma). The growth and colony-forming abilities of control cells were impaired with advancing age in vitro, especially at later stages (more than 70-80% of life span completed). It was found that treatment with TNF inhibited growth and colony-forming efficiency at any in vitro age. The effects of TNF were shown to increase with increasing in vitro age, as reflected by a more pronounced increase in doubling times, a decrease in saturation density, and a reduction in colony-forming efficiency. However, the characteristics of TNF receptors, including the dissociation constant, and the number of TNF-binding sites per cell-surface area remained rather constant. The effect of TNF was augmented by IFN-gamma at a dose that alone affected growth and colony formation only slightly. The augmentation by IFN-gamma was also found to depend on in vitro age; the synergy with TNF in the deterioration of colony-forming ability was observed only in "aged" cells. These results suggest that the intrinsic responsiveness of HUVECs to growth-inhibiting factors, as well as to growth-stimulating factors, changes during aging in vitro.

Studies on in vitro mechanisms of anti-tumor activity of the tumor necrosis factor alpha against human renal carcinoma cell line (KU-2).

To clarify the mechanisms of anti-tumor activity of human recombinant tumor necrosis factor alpha (rTNF alpha), an established cell line KU-2, derived from a patient with human renal cell carcinoma (RCC), was treated with rTNF alpha alone or in combination with anti-cancer agents: actinomycin-D (ACD), vinblastine sulfate (VLB), nimustine hydrochloride (ACNU), and methotrexate (MTX). Cytotoxic assay by crystal violet dye exclusion test showed that 21.0 +/- 4.0% and 34.8 +/- 4.7% of the cells were killed by 72 hours incubation with 100 ng/ml of rTNF alpha, alone and 1 ng/ml of ACD alone, respectively. Synergistic cytotoxicity of 75.0 +/- 0.3% was observed at 72 hours when 100 ng/ml of rTNF alpha and 1 ng/ml of ACD were added simultaneously. Furthermore, additive cytotoxicity of 48.5 +/- 1.1% was observed by 0.1 ng/ml of VLB and 100 ng/ml of rTNF alpha. However, when KU-2 was treated in conjunction with both 100 ng/ml of rTNF alpha and 3 micrograms/ml of ACNU or 2.5 ng/ml of MTX, no significant increase in cytotoxicity was demonstrated. When KU-2 was pretreated with 1 ng/ml of ACD for 24 hours, followed by adding 100 ng/ml of rTNF alpha, a synergistic cytotoxicity by ACD and rTNF alpha was observed at 24 hours. On the other hand, when KU-2 was pretreated with 100 ng/ml of rTNF alpha for 24 hours, followed by adding 1 ng/ml of ACD, no significant increase in cytotoxicity was demonstrated. In clonogenic assay studies, the colony forming efficiency (CE) of the control cultures was 31.8 +/- 8.1%. A 92.3 +/- 1.8% reduction in CE was observed when 100 ng/ml of rTNF alpha was added to the cultures. No significant synergistic or additive effects were demonstrated between rTNF alpha and chemotherapeutic agents in clonogenic assay studies. The effects of rTNF alpha on exponentially growing KU-2 cells were analyzed by studying the distribution of cells in the cell cycle. No cell cycle specific effect of rTNF alpha was demonstrated, regardless of whether or not chemotherapeutic agents were added. These results indicated that the cytotoxic and cytostatic activities of rTNF alpha may be mediated by separate mechanisms of action. Moreover, it was postulated that rTNF alpha may more significantly affect KU-2 cells having clonogenic potentials. rTNF alpha was concluded to have significant anti-tumor effects on renal cell carcinoma cells based on clonogenic assay studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of cis-diamminedichloroplatinum and mitomycin C-induced sister chromatid exchanges by dibutyryl cyclic AMP in a human salivary gland adenocarcinoma cell line in culture.

A human salivary gland adenocarcinoma cell line was cultivated in the presence of dibutyryl cyclic AMP (dB-cAMP), cis-diammine dichloroplatinum (cisplatin) or mitomycin C (MMC) only, or of the combination of dB-cAMP and each of the antineoplastic drugs. Then the treated cells were examined for the induction of sister chromatid exchanges (SCEs), colony-forming efficiency (CFE) in semisolid agar medium and cell survival as measured by counting for viable cells. It has been found that the frequency of SCE induction is significantly increased by treatment of cells with cisplatin or MMC under the presence of dB-cAMP, when compared to that in the cells treated with cisplatin or MMC only. Moreover, marked reduction in CFE and cell survival was found to occur in the cells treated with the combination of dB-cAMP and cisplatin or MMC. These finding indicate that treatment of a human salivary gland adenocarcinoma cell line with dB-cAMP results in modification of SCE induction, CFE and cell survival in the cells exposed to cisplatin or MMC.

Aromatase activity in human ovarian cancer

Eighty-four tumor samples from 70 women with primary ovarian cancer were assayed for cytosol estrogen (ERc) and progestin (PRc) receptor concentrations and aromatase activity. In addition, 22 of the tumors were studied for their response to the aromatase inhibitor, 4-OH-androstenedione, in a soft agar clonogenic cell assay system. Although aromatase activity was detected in almost all of the primary tumors, this enzyme was barely detectable in the majority of metastatic tumor samples. There was no significant correlation between aromatase activity and either the ERc or PRc content of the tumors, or tumor grade. Of 12 tumors grown successfully in the soft agar culture system, only 1 showed a substantial (>50%) reduction in colony-forming efficiency after exposure to the aromatase inhibitor. These results suggest that local estrogen biosynthesis probably does not play an important role in the majority of epithelial ovarian tumors. However, there may be a small subset of estrogen receptor-positive tumors in which aromatase could provide a local growth stimulus.

The toxic response of preneoplastic rat tracheal epithelial cells to 12-O-tetradecanoylphorbol-13-acetate.

The purpose of our studies was to examine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on survival, proliferation, and differentiation of normal and preneoplastic rat tracheal epithelial (RTE) cells. Normal RTE cells obtained from 8-week-old rats were plated into medium containing TPA at concentrations ranging from 0.1 to 100 ng/ml. A dose-related increase in colony-forming efficiency (CFE) was observed at concentrations above 0.1 ng/ml. At 100 ng/ml the CFE was increased 5-fold above that observed in controls. Similar studies were carried out with immortal, preneoplastic RTE cells derived from carcinogen-exposed primary cultures. Upon exposure to 10 ng/ml of TPA, 9 out of 11 cell lines showed a marked decrease in CFE ranging from a 70 to a greater than 99% reduction. Two out of 11 cell lines showed only a minor reduction in CFE at this TPA concentration. Teleocidin B was as potent as TPA in causing inhibition of CFE in preneoplastic RTE cells; the potency of 4-O-methyl TPA was approximately 1/100 that of TPA and phorbol was virtually inactive. Further studies suggested that inhibition of clonal growth of the preneoplastic tracheal cells by TPA was due to rapid cell death. As early as 2 h after addition of TPA to the cultures, a loss of viable cells from the cultures as well as a reduction in CFE was observed. Morphological studies support the interpretation that a large number of cells was severely damaged within 2--4 h after TPA exposure. The formation of cross-linked envelopes (CLE), a measure of keratinocyte differentiation, was determined in cultures of preneoplastic RTE cells. TPA caused only minor increases in CLE formation indicating that the cells were not dying from TPA-induced terminal keratinocyte differentiation, but rather from TPA toxicity. Thus, preneoplastic RTE cells respond differently to TPA from normal RTE cells and also from normal and transformed keratinocytes of various origins. The role of TPA toxicity in promoting preneoplastic RTE cells to the neoplastic state is being investigated.

Effectiveness of AMSA alone or in combination with radiation on murine fibrosarcoma pulmonary nodules

The cytotoxic effects in vivo of 4'-(9-acridinylamino) methanesulfon-m-anisidide (AMSA), radiation or both modalities in combination on murine fibrosarcoma (FSa) cells grown as pulmonary tumors were determined. Fourteen days following the i.v. injection of viable FSa cells, recipient mice developed between 100 and 150 visible pulmonary nodules. At that time, tumor-bearing animals were exposed to either single or combined modality treatments, as well as single and fractionated dose regimens. Animals were sacrificed 1 hour after the last treatment. Tumor nodules were excised, made into a single cell suspension and separated on the basis of cell size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the percentage contamination by normal diploid cells in each of the tumor cell populations. Known numbers of viable cells from each elutriator fraction were injected into recipient mice to determine their colony-forming efficiency (CFE). Surviving fractions were determined by comparing the CFEs of treated FSa cells from each of the separated elutriator fractions with those of appropriate untreated controls. Following a single i.v. dose of AMSA (30 mg/kg), populations of cells enriched in S phase were the most sensitive. When a single dose of AMSA was combined with a single dose of radiation (100 rad), there was a marked schedule dependence with the more effective sequence, especially if a 12 hour interval was chosen between doses, being AMSA followed by irradiation. No schedule dependence was observed if both modalities were combined and administered under a fractionated protocol of four fractions of AMSA (5 mg/kg per fraction) and four fractions of radiation (300 rad per fraction). Under these conditions the greatest reduction in CFE was in cell subpopulations most enriched in S and G2 + M phase cells.