Spontaneous Neoplastic Transformation Research Articles

In vitro evaluation of mesenchymal stromal cell senescence

Mesenchymal stromal cells (MSCs) are adult multipotent stem cells that, for their immunomodulatory and reparative/anti-inflammatory properties, are a promising tool in cell therapy. For the treatment of patients, MSCs are in vitro stimulated to proliferate in a non-physiological condition and this situation could lead MSCs to malignant transformation. The clinical application of MSCs requires that the biosafety of these cells must be investigated. The cellular senescence is a crucial mechanism that prevents the growth of cells at risk for neoplastic transformation. The aim of this study was to investigate the capacity of healthy donor bone marrow derived MSCs to enter the senescence phase by evaluating the β-galactosidase activity. We tested 131 MSC batches after long-term in vitro culture, resulting positive for β-gal staining and showing a branched shape morphology typical of senescent cells. They displayed a progressive decline in proliferative capacity and none of them bypassed the senescence. In conclusions, our data indicate that MSCs are not inclined towards spontaneous neoplastic transformation. We believe that the control of MSC capacity to enter senescence is fundamental for quality and safety, considering the relevant interest in the MSC clinical applications.

Abstract 5292: Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells

Abstract Obesity is a global epidemic with a predicted rate of 42% in the U.S.A by 2050. Epidemiologic studies show that obesity is a risk factor for developing cancer; however, the molecular mechanism has not been fully elucidated. Our published data demonstrate that fibroblast growth factor-2 (FGF2) released from fat cells (adipocytes) in the visceral adipose tissue (VAT) induces transformation/tumorigenicity in the skin and mammary epithelial cells. Specifically, FGF2 released from VAT stimulates epithelial cell growth in soft agar by inducing the proto-oncogene c-Myc. Growth in soft agar is a measure of transformation/tumorigenicity; neither transformation nor c-Myc induction in epithelial cells was reversible. c-Myc overexpression can initiate a process of genetic instability linked to tumor initiation. Our discovery of this novel direct path of VAT-stimulated tumorigenesis adds mechanistic insight to our earlier discovery that VAT secretions promote UVR-induced nonmelamoma skin cancer. The objective of our current study was to determine the mechanism by which FGF2 stimulates malignant transformation. We hypothesized that FGF2 from VAT induces c-Myc and subsequent genomic instability in epithelial cells, leading to increased carcinogenesis. To test hypothesis we generated a filtered conditioned-medium from the human VAT treated MCF-10A (mammary epithelial) and JB6 P+ (skin epithelial) cells and measured several downstream mediators of FGF2 and activation of FGFR-1 (FGF2 receptor). Following VAT treatment, epithelial cells demonstrated induced c-Myc protein expression along with ROS accumulation, elevated γ-H2AX foci, and increased micronucleus (MN) formation. We found that inhibition of c-Myc attenuated VAT-induced neoplastic transformation of MCF-10A and JB6 P+ cells, while constitutive activation of the c-Myc-induced spontaneous neoplastic transformation of JB6 P+ cells. Collectively, our data suggested FGF2 released from VAT interacts with FGFR-1 and activates c-Myc. The role of c-Myc in the formation of MN and DNA damage is under investigation. Determining the impact of excess VAT on cancer will lead to strategies to help prevent adiposity-associated cancers and identify individuals at risk for disease or individuals who may be susceptible to compounded genotoxicity due to DNA-damaging environmental exposures. Citation Format: Debrup Chakraborty, Vladislav Jdanov, Vanessa Benham, Blair Bullard, Jamie J. Bernard. Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5292.

A novel canine kidney cell line model for the evaluation of neoplastic development: karyotype evolution associated with spontaneous immortalization and tumorigenicity.

The molecular mechanisms underlying spontaneous neoplastic transformation in cultured mammalian cells remain poorly understood, confounding recognition of parallels with the biology of naturally occurring cancer. The broad use of tumorigenic canine cell lines as research tools, coupled with the accumulation of cytogenomic data from naturally occurring canine cancers, makes the domestic dog an ideal system in which to investigate these relationships. We developed a canine kidney cell line, CKB1-3T7, which allows prospective examination of the onset of spontaneous immortalization and tumorigenicity. We documented the accumulation of cytogenomic aberrations in CKB1-3T7 over 24 months in continuous culture. The majority of aberrations emerged in parallel with key phenotypic changes in cell morphology, growth kinetics, and tumor incidence and latency. Focal deletion of CDKN2A/B emerged first, preceding the onset and progression of tumorigenic potential, and progressed to a homozygous deletion across the cell population during extended culture. Interestingly, CKB1-3T7 demonstrated a tumorigenic phenotype in vivo prior to exhibiting loss of contact inhibition in vitro. We also performed the first genome-wide characterization of the canine tumorigenic cell line MDCK, which also exhibited CDKN2A/B deletion. MDCK and CKB1-3T7 cells shared several additional aberrations that we have reported previously as being highly recurrent in spontaneous canine cancers, many of which, as with CDKN2A/B deletion, are evolutionarily conserved in their human counterparts. The conservation of these molecular events across multiple species, in vitro and in vivo, despite their contrasting karyotypic architecture, is a powerful indicator of a common mechanism underlying emerging neoplastic activity. Through integrated cytogenomic and phenotypic characterization of serial passages of CKB1-3T7 from initiation to development of a tumorigenic phenotype, we present a robust and readily accessible model (to be made available through the American Type Culture Collection) of spontaneous neoplastic transformation that overcomes many of the limitations of earlier studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s10577-015-9474-8) contains supplementary material, which is available to authorized users.

Exposure to low level environmental agents: The induction of hormesis

Exposure to low level environmental agents: The induction of hormesis

Increased Frequency of Spontaneous Neoplastic Transformation in Progeny of Bystander Cells from Cultures Exposed to Densely Ionizing Radiation

An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons.

Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu

Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p < 30), and onset of aneuploidy. At high passage (p > 85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.

Suppression of Neoplastic Transformation in vitro by Low Doses of Low Let Radiation

A major concern of exposure to low doses of radiation is the risk of cancer induction. Epidemiologic data are rarely powerful enough to accurately discriminate this risk at doses <10 cGy. In order to gain insight into events at these low doses, laboratory-based studies of relevant endpoints are required. One such endpoint is radiation-induced neoplastic transformation in vitro. Such studies can provide quantitative dose-response data, as well as insights into underlying cellular and molecular mechanisms. Data are presented that indicate that low doses of low LET radiation can suppress neoplastic transformation in vitro to levels below those seen spontaneously. Mechanisms involved include both the death of a subpopulation of cells prone to spontaneous neoplastic transformation and the induction of DNA repair. The relative contributions of these mechanisms is dose-dependent. The relevance of these observations to radiation risk estimation is discussed.

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.

Radiation-induced neoplastic transformation in vitro: evidence for a protective effect at low doses of low LET radiation.

The study of radiation-induced transformation in vitro has long been an experimental approach to examine mechanisms underlying radiation carcinogenesis. Even though the major concern of exposure to radiation is the risk of cancer induction at low radiation doses, most laboratory mechanistic studies have focused on high dose effects. This, coupled with the fact that epidemiologic data are rarely powerful enough to accurately discriminate this risk at doses <5 cGy, has led in recent years to an increased effort to study low dose effects using the endpoint of neoplastic transformation in vitro. Since transformation frequencies at low doses are typically low (< 10(-4)), such studies are, by necessity, large and labor intensive. However, they have yielded quantitative dose-response data, as well as insights into underlying cellular and molecular mechanisms. An interesting, and potentially important, finding is that low doses of low LET radiation can suppress neoplastic transformation in vitro to levels below that seen spontaneously. Mechanistic studies have revealed that multiple mechanisms are likely to be involved, and these include both the death of a subpopulation of cells prone to spontaneous neoplastic transformation and the induction of DNA repair. The relative contribution of these mechanisms appears to be dose-dependent. The relevance of in vitro studies to carcinogenesis in vivo is discussed.

Spontaneous Neoplastic Transformation of the Gill Cell Line FG-9307 from the Olive FlounderParalichthys olivaceus

Abstract The present study reports that FG-9307 cells derived from the gills of the olive flounder Paralichthys olivaceus underwent neoplastic transformation spontaneously during the process of in vitro culture. Cells lost their density-dependent inhibition and contact inhibition properties, and they acquired genomic variation and the capacity of piling up in a focus and growing in semisolid agar. This is the first report of in vitro spontaneous neoplastic transformation of fish cell lines. The preneoplastically and neoplastically transformed FG cells provide a powerful tool to screen carcinogenic and anticancer agents in the aquatic environment.

Explanation of protective effects of low doses of γ -radiation with a mechanistic radiobiological model

Purpose : To test whether data that show protective effects of low doses against spontaneous neoplastic transformation of C3H 10T1/2 cells can be explained with a biomathematical model that includes radioprotective mechanisms. To link important features of the model to known biological processes. Materials and methods : The model simulates double-strand break formation in transcriptionally active and in bulk DNA, translocation of DNA segments, and the fixation of damage at mitosis; promotion is also included. The model equations were solved numerically using a stiff solver. Results : The data were successfully simulated by the model: cell transformation-reducing effects of low doses of γ-radiation delivered at low dose-rates are explained by radiation-inducible DNA repair and enzymatic scavenging. Conclusions : The model successfully simulates experimental data. The highly nonlinear features of the data point to a nonlinear dose-effect relationship at low doses and indicate that linear extrapolation from moderate (or high) to low doses and dose-rates may not be justified for in vitro studies of the cell line under consideration.

The shape of the dose-response curve for radiation-induced neoplastic transformation in vitro: evidence for an adaptive response against neoplastic transformation at low doses of low-LET radiation.

A dose-response curve for gamma-radiation-induced neoplastic transformation of HeLa x skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with (137)Cs gamma radiation and either plated immediately or held for 24 h at 37 degrees C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2(n), where n is the number of observations (5); i.e., 1/32 congruent with 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of gamma radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.

Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse.

The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27(KIP1) expression have been detected in most advanced prostate cancers. But mice deficient for Cdkn1b (encoding p27(Kip1)) do not develop prostate cancer. PTEN activity leads to the induction of p27(KIP1) expression, which in turn can negatively regulate the transition through the cell cycle. Thus, the inactivation of p27(KIP1) may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten(+/-)/Cdkn1b(-/-) mice. Moreover, Pten(+/-)/Cdkn1b(-/-) mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27(Kip1) through the control of cell-cycle progression.

Spontaneous Neoplastic Transformation of WB-F344 Rat Liver Epithelial Cells

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.

Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells

The genomic evolution of a cohort of WB-F344 rat liver epithelial cell lineages undergoing spontaneous neoplastic transformation was followed to define the mechanistic relationship between genomic instability and progression to the neoplastic phenotype. Eighteen independent populations of WB-F344 cells (initiated from a single diploid-founding population) were subjected to 12 cycles of selective growth at confluent cell density, and cellular DNA contents were measured after each selection cycle. Flow cytometry demonstrated significant gains in the amount of G1 DNA after selection cycles 3, 6, and 7 in 44% (8 of 18), 89% (16 of 18), and 39% (7 of 18) of the cell populations, respectively. All populations subsequently lost DNA and returned to a diploid or pseudo-diploid DNA content within 1 to 2 selection cycles after the appearance of an increased DNA content. Additionally, appearance and subsequent disappearance of aneuploid or tetraploid subpopulations was observed in 11% (2 of 18) and 83% (15 of 18) of the experimental lineages, respectively. Although perturbations of G1 DNA content were apparent as early as selection cycle 3, at least 8 cycles of selective growth were required for the acquisition of tumorigenicity. While the independent lineages demonstrated significant fluctuations in G1 DNA content between selection cycles 3 and 8, the majority (11 of 13) of the populations contained a diploid or pseudo-diploid DNA content at the time tumorigenicity was expressed. Genomic instability preceded the acquisition of tumorigenic potential in rat liver epithelial cells subjected to selective growth conditions of maintenance at confluence, and may be required for its expression.

Ku70: A Candidate Tumor Suppressor Gene for Murine T Cell Lymphoma

We present evidence that inactivation of the Ku70 gene leads to a propensity for malignant transformation both in vitro and in vivo. In vitro, Ku70 −/− mouse fibroblasts displayed an increased rate of sister chromatid exchange and a high frequency of spontaneous neoplastic transformation. In vivo, Ku70 −/− mice, known to be defective in B but not T lymphocyte maturation, developed thymic and disseminated T cell lymphomas at a mean age of 6 months with CD4 +CD8 + tumor cells. These findings directly demonstrate that Ku70 deficiency facilitates neoplastic growth and suggest a novel role of the Ku70 locus in tumor suppression.

Induction of an Adaptive Response against Spontaneous Neoplastic Transformation In Vitro by Low-Dose Gamma Radiation

An adaptive response against spontaneous neoplastic transformation in vitro induced by low-dose gamma radiation has recently been reported using a clone of C3H 10T1/2 cells with a predisposition toward spontaneous transformation (Azzam et al., Radiat. Res. 146, 369-370, 1996). To test the generality of this observation, the HeLa x skin fibroblast human hybrid cell system was used to look for such an adaptive response using a similar experimental protocol. In the experimental protocol used, the frequency of neoplastic transformation of unirradiated cultures (Arm A) was compared to that of cultures which had been irradiated with 1 cGy of gamma radiation and either plated immediately (Arm B) or held for 24 h at 37 degrees C prior to plating (Arm C) for assay of neoplastic transformation. The pooled data from four separate experiments demonstrated a significantly reduced transformation frequency for Arm C compared to Arms A and B. This is indicative of an adaptive response in the case of postirradiation holding, in agreement with the earlier study using C3H 10T1/2 cells. However, with the exception of one experiment, the existence of the adaptive response was not clear on the basis of analysis of individual experiments. This points out the importance of sample size when looking at low-dose effects that are close in magnitude to the inherent variations in spontaneous transformation frequency.

Spontaneous Neoplastic Transformation of WB-F344 Rat Liver Epithelial Cells

Spontaneous Neoplastic Transformation of WB-F344 Rat Liver Epithelial Cells

Sensitivity of transformation to small differences in population density during serial passage of NIH 3T3 cells.

Early passages of the NIH 3T3 mouse cell line undergo spontaneous neoplastic transformation leading to the development of transformed foci if grown to confluence in 2% (vol/vol) calf serum (CS) and left there for more than a week. Transfer of the postconfluent cultures results in the appearance of large numbers of transformed foci; many of them are larger and denser than those in the original culture. If the cells are continually kept at low population densities by frequent passages in 10% CS, they lose the capacity to undergo spontaneous transformation. If however the low-density passages are made in 2% CS or in 10% (vol/vol) fetal bovine serum, both of which support lower growth rates and saturation densities than does 10% CS, they gain the capacities to grow to high saturation densities and produce more foci when grown to confluence in 2% CS. These increases are proportional to the population densities used in the frequent passages, although the densities are all kept well below confluence. We conclude that the combined constraints of submaximal serum plus those of the limited cell contacts of the low cell densities used here elicit an adaptive response that endows the entire population with increased growth capacity. The increased growth capacity of the heterogeneous population in turn increases the capacity of a fraction of the population to initiate distinctive transformed foci. Similar studies have indicated that the capacity of cells to produce tumors and metastases in mice and rats is enhanced by prior maintenance at high density in culture. We propose the concept of progressive state selection to account for the general increase in the growth capacity of cells that is elicited by moderate constraints on their growth and metabolism.

A free-radical hypothesis for the instability and evolution of genotype and phenotype in vitro.

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., an in vitro evolution, which includes crisis, spontaneous transformation ('immortalization'), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by the in vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause of in vitro evolution. We propose an oxyradical hypothesis to explain each of the stages of in vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.