Event In Cell Transformation Research Articles

LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects

Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.

Enhanced morphological transformation of human lung epithelial cells by continuous exposure to cellulose nanocrystals

Cellulose nanocrystals (CNC), also known as nanowhiskers, have recently gained much attention due to their biodegradable nature, advantageous chemical and mechanical properties, economic value and renewability thus making them attractive for a wide range of applications. However, before these materials can be considered for potential uses, investigation of their toxicity is prudent. Although CNC exposures are associated with pulmonary inflammation and damage as well as oxidative stress responses and genotoxicity in vivo, studies evaluating cell transformation or tumorigenic potential of CNC’s were not previously conducted. In this study, we aimed to assess the neoplastic-like transformation potential of two forms of CNC derived from wood (powder and gel) in human pulmonary epithelial cells (BEAS-2B) in comparison to fibrous tremolite (TF), known to induce lung cancer. Short-term exposure to CNC or TF induced intracellular ROS increase and DNA damage while long-term exposure resulted in neoplastic-like transformation demonstrated by increased cell proliferation, anchorage-independent growth, migration and invasion. The increased proliferative responses were also in-agreement with observed levels of pro-inflammatory cytokines. Based on the hierarchical clustering analysis (HCA) of the inflammatory cytokine responses, CNC powder was segregated from the control and CNC-gel samples. This suggests that CNC may have the ability to influence neoplastic-like transformation events in pulmonary epithelial cells and that such effects are dependent on the type/form of CNC. Further studies focusing on determining and understanding molecular mechanisms underlying potential CNC cell transformation events and their likelihood to induce tumorigenic effects in vivo are highly warranted.

Methylation Dynamics of RASSF1A and Its Impact on Cancer.

5-methyl cytosine (5mC) is a key epigenetic mark entwined with gene expression and the specification of cellular phenotypes. Its distribution around gene promoters sets a barrier for transcriptional enhancers or inhibitor proteins binding to their target sequences. As a result, an additional level of regulation is added to the signals that organize the access to the chromatin and its structural components. The tumor suppressor gene RASSF1A is a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway. RASSF1A action stimulates mitotic arrest, DNA repair and apoptosis, and controls the cell cycle and cell migration. De novo methylation of the RASSF1A promoter has received much attention due to its increased frequency in most cancer types. RASSF1A methylation is preceded by histones modifications and could represent an early molecular event in cell transformation. Accordingly, RASSF1A methylation is proposed as an epigenetic candidate marker in many cancer types, even though an inverse correlation of methylation and expression remains to be fully ascertained. Some findings indicate that the epigenetic abrogation of RASSF1A can promote the alternative expression of the putative oncogenic isoform RASSF1C. Understanding the complexity and significance of RASSF1A methylation is instrumental for a more accurate determination of its biological and clinical role. The review covers the molecular events implicated in RASSF1A methylation and gene silencing and provides a deeper view into the significance of the RASSF1A methylation patterns in a number of gastrointestinal cancer types.

Human papilloma virus infection and fascin over-expression in squamous cell carcinoma of the cervix.

Background: Human papilloma virus (HPV) is involved in development of almost all cervical cancers, mainly through the subversion of cellular mechanisms of growth control. Fascin plays central role in subsequent cell transformation events. Fascin mediates stabilization of parallel actin bundles where cellular protrusions are formed; this represents primary stages of cell migration and metastasis. Immunohistochemical assays have shown up-regulation of fascin expression in many epithelial and non-epithelial neoplasms. Therefore, the aim of this study was to investigate HPV infection and fascin expression in samples of cervical cancer. Methods: Of 66 patients with confirmed SCC, formalin-fixed specimens, embedded in paraffin blocks were evaluated for HPV infection with nested multiplex polymerase chain reaction (NM-PCR) and for fascin expression with immunohistochemical assays. Statistical analysis was performed using Wilcoxon rank-sum test and SPSS software. A p<0.05 was considered for statistical significance. Results: Of 66 samples, 52 (78.7%) were found positive for HPV infection and fascin over-expression was shown in all squamous cell carcinoma samples. Conclusion: This study showed fascin overexpression in squamous cell carcinoma of the cervix which might be involved in metastasis of cancers induced by some types of HPV, hypothetically through attenuation of inter-cellular adhesions, and induction of cell motility

Dimensions of DNA Damage Recognition are Key Events in Inducing Complementary Multi-Pathway Repair Response in Potentiating Tumor Therapeutic Resistance

Dimensional resolution of malignant transformation events in cells is a putative reflection of susceptibility hot spots in DNA biology of response to genomic damage. It is further to such understanding that repair processes are potentially a vast array of complementary pathways that significantly promote the resistance to therapeutic attempts at cancerous lesion ablation. Sufficient promotional repair of single-strand and double-strand breaks may subsequently compromise susceptibility to therapy once cell replication is initiated. The toxic nature of abasic sites is a prime example for a need for selective phase susceptibility during the cell cycling of tumor cells to engineered or natural chemotherapeutic agents or to ionizing radiation.

Effect of surface functionalizations of multi-walled carbon nanotubes on neoplastic transformation potential in primary human lung epithelial cells

Functionalized multi-walled carbon nanotube (fMWCNT) development has been intensified to improve their surface activity for numerous applications, and potentially reduce toxic effects. Although MWCNT exposures are associated with lung tumorigenesis in vivo, adverse responses associated with exposure to different fMWCNTs in human lung epithelium are presently unknown. This study hypothesized that different plasma-coating functional groups determine MWCNT neoplastic transformation potential. Using our established model, human primary small airway epithelial cells (pSAECs) were continuously exposed for 8 and 12 weeks at 0.06 μg/cm2 to three-month aged as-prepared-(pMWCNT), carboxylated-(MW-COOH), and aminated-MWCNTs (MW-NHx). Ultrafine carbon black (UFCB) and crocidolite asbestos (ASB) served as particle controls. fMWCNTs were characterized during storage, and exposed cells were assessed for several established cancer cell hallmarks. Characterization analyses conducted at 0 and 2 months of aging detected a loss of surface functional groups over time due to atmospheric oxidation, with MW-NHx possessing less oxygen and greater lung surfactant binding affinity. Following 8 weeks of exposure, all fMWCNT-exposed cells exhibited significant increased proliferation compared to controls at 7 d post-treatment, while UFCB- and ASB-exposed cells did not differ significantly from controls. UFCB, pMWCNT, and MW-COOH exposure stimulated significant transient invasion behavior. Conversely, aged MW-NHx-exposed cells displayed moderate increases in soft agar colony formation and morphological transformation potential, while UFCB cells showed a minimal effect compared to all other treatments. In summary, surface properties of aged fMWCNTs can impact cell transformation events in vitro following continuous, occupationally relevant exposures.

ETV6/RUNX1 transcript is a target of RNA-binding protein IGF2BP1 in t(12;21)(p13;q22)-positive acute lymphoblastic leukemia

The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) is overexpressed in a subset of cancers and promotes cell cycle, migration and aggressive phenotype by regulating post-transcriptionally a number of key mRNAs (e. g, ACTB, CD44, CTNNB1, KRAS, MAPK4, MYC, PTEN and others). IGF2BP1 is also overexpressed in t(12;21)(p13;q22)-positive acute lymphoblastic leukemia (ALL), but the biological significance of this phenomenon has not been addressed so far. We have identified leukemia fusion gene ETV6/RUNX1 mRNA to be highly enriched in immunoprecipitated fraction of endogenous IGF2BP1 from a model cell line REH and t(12;21)(p13;q22)-positive ALL samples. Furthermore, downregulation of IGF2BP1 by two-fold has resulted in a corresponding decrease of ETV6/RUNX1 mRNA validating this transcript as a target of IGF2BP1 protein in t(12;21)(p13;q22)-positive ALL.These data infer that IGF2BP1 is a potent regulator of ETV6/RUNX1 mRNA stability and potentially link this evolutionary-highly conserved protein to cell transformation events in ETV6/RUNX1-mediated leukemogenesis of t(12;21)(p13;q22)-positive ALL.

Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia

Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development.

KSRP silencing favors neural differentiation of P19 teratocarcinoma cells

Understanding the molecular mechanisms that control the balance between multipotency and differentiation is of great importance to elucidate the genesis of both developmental disorders and cell transformation events. To investigate the role of the RNA binding protein KSRP in controlling neural differentiation, we used the P19 embryonal carcinoma cell line that is able to differentiate into neuron-like cells under appropriate culture conditions. We have recently reported that KSRP controls the differentiative fate of multipotent mesenchymal cells owing to its ability to promote decay of unstable transcripts and to favor maturation of selected micro-RNAs (miRNAs) from precursors. Here we report that KSRP silencing in P19 cells favors neural differentiation increasing the expression of neuronal markers. Further, the expression of two master transcriptional regulators of neurogenesis, ASCL1 and JMJD3, was enhanced while the maturation of miR-200 family members from precursors was impaired in KSRP knockdown cells. These molecular changes can contribute to the reshaping of P19 cells transcriptome that follows KSRP silencing. Our data suggests that KSRP function is required to maintain P19 cells in a multipotent undifferentiated state and that its inactivation can orient cells towards neural differentiation.

Roles of SM22α in Cellular Plasticity and Vascular Diseases

SM22α is a shape change and transformation sensitive 22 kDa actin-binding protein of the calponin family. It is ubiquitous to vascular and visceral smooth muscle, and is an early marker of smooth muscle differentiation. It is also present in fibroblasts, and some epithelium. SM22α may be involved in calcium-independent smooth muscle contraction. Recent evidence suggests that disruption of SM22α induces vascular inflammation and is involved in osteochondrogenesis in arterial diseases. This is consistent with activation of NF-κB signaling, where NF-κB activity is upregulated in vascular injury. High expression of SM22α inhibits cell proliferation in VSMCs and in injured arteries. SM22α acts as a tumor suppressor. Loss of its expression is an early event in cell transformation and the development of some tumors, coinciding with cellular plasticity.

3D-modelling of radon-induced cellular radiobiological effects in bronchial airway bifurcations: Direct versus bystander effects

Purpose: The primary objective of this paper was to investigate the distribution of radiation doses and the related biological responses in cells of a central airway bifurcation of the human lung of a hypothetical worker of the New Mexico uranium mines during approximately 12 hours of exposure to short-lived radon progenies. Materials and methods: State-of-the-art computational modelling techniques were applied to simulate the relevant biophysical and biological processes in a central human airway bifurcation. Results: The non-uniform deposition pattern of inhaled radon daughters caused a non-uniform distribution of energy deposition among cells, and of related cell inactivation and cell transformation probabilities. When damage propagation via bystander signalling was assessed, it produced more cell killing and cell transformation events than did direct effects. If bystander signalling was considered, variations of the average probabilities of cell killing and cell transformation were supra-linear over time. Conclusions: Our results are very sensitive to the radiobiological parameters, derived from in vitro experiments (e.g., range of bystander signalling), applied in this work and suggest that these parameters may not be directly applicable to realistic three-dimensional (3D) epithelium models.

Escape from p21-mediated Oncogene-induced Senescence Leads to Cell Dedifferentiation and Dependence on Anti-apoptotic Bcl-xL and MCL1 Proteins

Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.

Effect of site-specific bronchial radon progeny deposition on the spatial and temporal distributions of cellular responses

Inhaled short-lived radon progenies may deposit in bronchial airways and interact with the epithelium by the emission of alpha particles. Simulation of the related radiobiological effects requires the knowledge of space and time distributions of alpha particle hits and biological endpoints. Present modelling efforts include simulation of radioaerosol deposition patterns in a central bronchial airway bifurcation, modelling of human bronchial epithelium, generation of alpha particle tracks, and computation of spatio-temporal distributions of cell nucleus hits, cell killing and cell transformation events. Simulation results indicate that the preferential radionuclide deposition at carinal ridges plays an important role in the space and time evolution of the biological events. While multiple hits are generally rare for low cumulative exposures, their probability may be quite high at the carinal ridges of the airway bifurcations. Likewise, cell killing and transformation events also occur with higher probability in this area. In the case of uniform surface activities, successive hits as well as cell killing and transformation events within a restricted area (say 0.5mm(2)) are well separated in time. However, in the case of realistic inhomogeneous deposition, they occur more frequently within the mean cycle time of cells located at the carinal ridge even at low cumulative doses. The site-specificity of radionuclide deposition impacts not only on direct, but also on non-targeted radiobiological effects due to intercellular communication. Incorporation of present results into mechanistic models of carcinogenesis may provide useful information concerning the dose-effect relationship in the low-dose range.

Abstract P2-09-34: ERK Phosphorylation in Fibroblasts within Breast Cancer Stroma Is Associated with an Improved Tamoxifen Response

Abstract Background. Extra-cellular signal-regulated kinase (ERK) 1/2 is an integral part of the mitogen-activated protein kinase (MAPK) pathway, transmitting signals involved in cell proliferation and cell death upon binding of extracellular growth factors to their receptors. Whereas its role in tumour cells has been linked to key events in cell transformation, its role in tumour-associated fibroblasts, a component of the tumour stroma, is largely unknown. Material and methods. Tissue microarrays (TMAs) of two cohorts including invasive breast cancer patients were stained for ERK phosphorylation level (pERK) and SMA-alpha expression (SMAa) which were scored and analysed in regard to clinico-pathological, prognostic and molecular markers and recurrence-free survival. Results. In both clinical materials we show a significant correlation between stromal ERK phosphorylation and the marker for activated fibroblasts SMAa. SMAa expression was not linked to treatment-predictive information but instead had prognostic qualities (P=0.034, multivariate Cox regression). By analysing the unique randomised tamoxifen trial including patients receiving no adjuvant treatment we show for the first time that ERK phosphorylation in cancer-associated fibroblasts (CAF) was associated with an improved outcome upon tamoxifen treatment. In contrast, patients having ERα-positive tumours but lacking ERK phosphorylation in the stroma did not respond to tamoxifen treatment (P=0.015, multivariate interaction analyses). Discussion. This suggests that only a subpopulation of activated fibroblasts, defined by phosphorylation of ERK, is linked to a response to tamoxifen treatment illustrating the importance of the stroma for monitoring and potentially mediating treatment effects in premenopausal breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-34.

Progesterone induces adult mammary stem cell expansion

Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer.

Spontaneous cell transformation: karyoplasts derived from multinucleated cells produce new cell growth in senescent human epithelial cell cultures.

Previously, it was shown that SV40-induced cell transformation of human diploid (2N), epithelial cells was a dynamic process of nuclear and cellular events. In this process, nuclei of polyploid (above 2N) cells broke down into multinucleated cells (MNCs) by amitotic division. An induced mass karyoplast (i.e., small cell with reduced amount of cytoplasm) budding process from the MNCs produced transformed cells with extended life span (EL) and altered morphology. In this study, without the use of SV40 and no induction of karyoplast budding, the same sequence of cellular events was found to occur spontaneously for the same type of cells at replicative senescence (no mitosis). These cell transformation events were followed by phase-contrast photography of living cell cultures. Primary, diploid, epithelial cell cultures grew for two to three passages and then entered senescence. Cells remaining in the cultures after widespread cell death (mortality stage 1; M1) developed the typical large, flat-cell morphology of senescence with increased cytoplasmic volume. Some of these cells were MNCs, mostly with two to four nuclei. Cytokinesis in MNCs and spontaneous karyoplast budding from MNCs were observed, and new, limited EL cell growth was present either in foci of cells or as prolonged cell growth over one to two passages. At the end of their replicative phase, the EL cells entered another death crisis (M2) from which no cells survived. In M2-crisis, rarely transformed cells appear with immortal cell growth characteristics (i.e., cell lines). Numerous examples of fragmentation or amitosis of polyploid nuclei in the production of multinucleated cells (MNCs) are presented. Such nuclear divisions produced nuclei with unequal sizes, which suggest unbalanced chromosomal segregations. The nuclear and cellular events in cell transformation are compared with a natural (no induction) occurrence of MNC-offspring cells in mammalian placentas. The possibility of a connection between these two processes is discussed. And finally the difference in the duration of EL cell growth from SV40-MNCs versus from senescent-MNCs is ascribed to increased mutational load in SV40-induced MNCs as compared with that in senescence MNCs.

Parallel patterns of cell-specific gene expression during enterocyte differentiation and maturation in the small intestine of the rabbit

Enterocytes are the major epithelial cell type of the small intestine. Their capacity to secrete, absorb and digest specific ions and nutrients is dependent on their position along the length of the small intestine as well as their stage of development as they migrate and differentiate along the crypt-villus axis. In order to further understand the molecular processes that regulate enterocyte differentiation and function, this study has compared the levels of six mRNA species produced by genes expressed in rabbit enterocytes; specifically, the multidrug resistance ( MDR1) gene encoding the 170-kDa P-glycoprotein, CaBP 9k, which encodes a putative intracellular calcium buffer, calbindin, LPH, APN, and AP which encode the brush-border hydrolases lactase-phlorizin hydrolase, aminopeptidase N and alkaline phosphatase, respectively, and SGLT1, encoding the brush border Na +-glucose cotransporter. The level of each mRNA species has been mapped along the small intestine using quantitative in situ hybridisation. This has revealed characteristic regional variations in the abundance of each of the mRNAs, supporting the opinion that there is a strong genetic component to the maintenance of gradients in epithelial function along the length of the small intestine. Analysis of the cellular accumulation of mRNA during enterocyte migration along the crypt-villus axis, over gut-associated lymphoid tissue, and at epithelial boundaries, has, by contrast, established a clear correlation in the expression of these genes. These data illustrate the dynamics of enterocyte gene expression, thereby providing an insight into the molecular mechanisms which co-ordinate the events of cell transformation that underlie functional differences between the epithelial populations of the small intestine.

High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer.

DNA methylation abnormalities occur consistently in human neoplasia including widespread hypomethylation and more recently recognized local increases in DNA methylation that hold potential for gene inactivation events. To study this imbalance further, we have cloned and localized to chromosome 19 a portion of the human DNA methyltransferase gene that codes for the enzyme catalyzing DNA methylation. Expression of this gene is low in normal human cells, significantly increased (30- to 50-fold by PCR analysis) in virally transformed cells, and strikingly elevated in human cancer cells (several hundredfold). In comparison to colon mucosa from patients without neoplasia, median levels of DNA methyltransferase transcripts are 15-fold increased in histologically normal mucosa from patients with cancers or the benign polyps that can precede cancers, 60-fold increased in the premalignant polyps, and greater than 200-fold increased in the cancers. Thus, increases in DNA methyltransferase gene expression precede development of colonic neoplasia and continue during progression of colonic neoplasms. These increases may play a role in the genetic instability of cancer and mark early events in cell transformation.

Identification of Transformation-Related Antigen by Monoclonal Antibody on Swiss 3T3 Cells Induced by Transfection With Murine Cultured Colon 36 Tumor DNA2

The cell surface antigen associated with the transformed state of cells that could grow in an anchorage-independent manner was analyzed by use of techniques of DNA transfection and hybridomas secreting the monoclonal antibody (MoAb). Spleen cells of C57BL/6 mice immunized with a highly tumorigenic, chemically induced murine cultured colon 36 tumor (C-C36) of BALB/c origin were hybridized with NS-1, a hypoxanthine phosphoribosyltransferase-deficient myeloma line of BALB/c mice. Screening of hybridomas revealed an antibody that reacted with C-C36 and transformed Swiss 3T3 cells growing in soft agar after transfection of 3T3 cells with C-C36 DNA. The hybridomas that did not react with nontransformed 3T3 and the less tumorigenic BALB/c hemangioendothelioma line D10 were then selected. An MoAb was designated "#71295." This MoAb immunoprecipitated the antigen that consisted of 65,000- and 14,000-molecular-weight components with soluble C-C36 membrane antigens. It also reacted with 2 other chemically induced syngeneic colon tumor lines, cultured colon 26 tumor line and cultured colon 51 tumor line, and with fibrosarcoma Meth A. However, #71295 was not found in NS-1, D14, and BALB/c normal thymus, liver, colon, and kidney tissues. In addition, this MoAb could not inhibit the anchorage-independent growth of C-C36 and transformed 3T3 cells. These results suggest that although the molecule defined by #71295 might not be associated with the anchorage independence of cell growth, it could be a newly expressed determinant on the cell surface that is related to the events of cell transformation.