Colony-forming Ability In Soft Agar Research Articles

Malignant Progression of Human CML Clone Demonstrates a Unique Developmental Pathway Characterized by Altered Expression Pattern of Cancer, Adhesion and Drug Resistance Genes.

The K562 cell line is a clone derived from a patient with CML in blast crisis and is widely used as a meaningful model for experimental and clinical studies. Similarly to primary CML blasts, K562 grow mostly in suspension in the absence of supportive bone marrow stromal cells or plate-coating of adhesion molecules. However, under standard culture conditions a relatively small cell-fraction (2–3%) adheres to the plastic dish. In this study, we investigated whether the plastic-adhesive sub-population is a side population of clonal origin and whether it has unique malignant properties. For these reasons, we have established and characterized a new cellular model system of plastic-adhesive variants of K562 (termed K562/Adh and K562/sAdh). These adhesive-variants retain self-renewal ability with indications of increased malignant properties as increased colony-forming ability in soft agar, impairment of contact inhibition and loss of serum-dependent attenuation of adhesion. cDNA microarray analyses that were validated by quantitative RT-PCR analyses have shown altered expression levels of adhesion related genes in the adhesive variants relatively to the parental cells. This included 4.0-, 3.1- and 2.5- fold up-regulation of the KiSS-1, THBS3, ITGB5 genes, respectively, and 1.7- fold down-regulation of the FN1 gene. Moreover, up-regulation of the BCR-ABL fusion gene (3.1- fold) and of the AP1 transcription factors genes, c-JUN (4.5-fold) and c-FOS (2.8-fold), was found in the adhesive variants relatively to their parental cells. Other transcription factors from the AP1 family (FRA1, JUN-B and JUN-D) similarly expressed in all K562 variants. Interestingly, both adhesive cell variants also demonstrated a de-novo expression of the multidrug resistance, MDR1 gene. Our data strongly suggest that the adhesive-variants of K562 cells are an outcome of a unique pathway of malignant progression that not only involves novel adhesion properties. Cellular cloning experiments and molecular gene-expression analyses of various clones indicated that this malignancy progression pathway is a global inherited property of K562 cells. Although the progeny of any parental K562 have the potential to transform into adherent cells, only a confined fraction (3.0±0.5 %) of progeny cells undergo such transformation in apparently stochastic manner. The factors involved in the induction of this transformation process in a particular progeny cell are yet unknown. However, growing K562 cells in pre-conditioned medium from cultures of K562/Adh or K562/sAdh did not change the size of adhered cell fraction, suggesting that their adhesion is independent of molecules that are specifically released or secreted by the adhesive variants of K562. Neither growing K562/Adh nor K562/sAdh in pre-conditioned medium from cultures of the parental K562 affected their adhesion properties. In conclusion, we demonstrated existence of a unique unidirectional, adhesion-based transformation pathway of CML blasts. This pathway is also characterized by altered pattern of gene expression that is associated with increased malignant properties. As the resultant transformed cells can be discriminated by adhesion to plastic, this system may serve as a model to investigate the clinical and therapeutic implications of such transformation in CML progression, drug-resistance and minimal residual disease.

Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies.

The alpha-folate receptor (FR) is selectively overexpressed in 90% of nonmucinous ovarian carcinomas, whereas no expression is detectable in normal ovarian surface epithelium (OSE). Indirect evidence suggests that FR expression is associated with tumor progression and affects cell proliferation. To evaluate better the role of FR, we developed an approach based on intracellular expression of single-chain (sc) antibodies (intrabody) to downmodulate membrane expression of FR in ovary cancer cells. IGROV-1 and SKOV3 ovarian carcinoma cell lines were transfected with an anti-FR intrabody. Transfectants and parental cells were tested for FR, integrins and anti-FR intrabody expression by fluorescence-activated cell sorting (FACS), reverse transcription and polymerase chain reaction (RT-PCR) and/or immunoblotting. Cell growth characteristics and adhesion properties were evaluated in liquid, semisolid and organotypic cultures. The anti-FR scFv inhibited FR expression from 60 to 99%. At physiological concentrations of folate, proliferation varied directly as a function of FR expression. FR downmodulation was accompanied by reduced colony-forming ability in soft agar, morphological change of the cells, significant enhanced adhesion to laminin or Matrigel, a two- to three-fold increase in alpha6beta4 integrin expression, and a marked reduction in laminin production. In three-dimensional organotypic cultures, anti-FR intrabody-transfected IGROV1 cells grew as a single-ordered layer, reminiscent of normal OSE growth in vivo. In conclusion, the anti-FR intrabody reverses the transformed phenotype in ovary cancer cells and may provide an efficient means to inhibit selectively the growth of these cells.

Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth, apoptosis, and gene expression in3LL Lewis lung carcinoma cells

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and beta1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF alpha receptor mRNA and protein, but in the SM3-reconstituted cells PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.

Antisense downregulation of N-myc1 in woodchuck hepatoma cells reverses the malignant phenotype.

Cell line WH44KA is a highly malignant woodchuck hepatoma cell line. WH44KA cells contain a single woodchuck hepatitis virus (WHV) DNA integration in the 3' untranslated region of exon 3 of the woodchuck N-myc1 gene. The highly rearranged WHV DNA contains WHV enhancers which activate the N-myc promoter, and a hybrid N-myc1-WHV mRNA is produced, which leads to a high steady-state level of N-myc1 protein. To investigate whether continuous N-myc1 expression is required to maintain the tumor phenotype, we knocked out N-myc expression using a WHV-N-myc1 antisense vector. We identified two WH44KA antisense cell lines, designated 4-5 and 4-11, in which steady-state N-mycl protein levels were reduced by 95 and 80%, respectively. The growth rates of both antisense cell lines were reduced in comparison to those of wild-type and vector controls. The phenotype of 4-5 and 4-11 cells changed to a flattened appearance, and the cells exhibited contact inhibition. Colony-forming ability in soft agar was reduced by 92% for 4-5 cells and by 88% for 4-11 cells. Cell line 4-11 formed only small, slow-growing tumors in nude mice, consistent with a low level of N-myc1 remaining in the cells. In contrast, 4-5 cells, in which N-myc protein was reduced by greater than 95%, failed to form tumors in nude mice. The integrated WHV DNA contained the complete WHV X gene (WHx) and its promoter; however, we did not detect any WHx protein in the cells by using a sensitive assay. These data demonstrate that N-myc overexpression is required to maintain the malignant phenotype of WH44KA woodchuck hepatoma cells and provide a direct function for integrated WHV DNA in hepatocarcinogenesis.

Transfection of IFNalpha in human glioblastoma cells and tumorigenicity in association with induction of PKR and OAS gene expression.

Previously these authors and others demonstrated frequent homozygous deletions of the chromosome 9p-localized class I interferon (IFN) gene cluster in glioblastoma tumors and cell lines. To investigate the biological effects of class I IFN gene transfer and constitutive expression in glioblastoma cells devoid of this gene cluster, the authors have developed a stable IFNalpha "transfectant" of the cell line U118. The expression of IFNalpha protein in the U118 transfectant clone is associated with decreased levels of DNA synthesis exhibited by cultures of transfected cells, reduced colony-forming ability in soft agar, and loss of tumorigenicity in athymic nude mice. To address the molecular consequences of constitutive IFNalpha synthesis, they examined the expression of four genes whose transcription has been shown to be responsive to IFN-mediated signal transduction and could be important to the observed antiproliferative and antitumor effects. Northern blot analysis revealed that changes in the levels of messenger (m)RNA for two of these genes, c-myc and mhc class I, are minor. However, mRNAs for oligoadenylate synthetase (OAS) as well as double-stranded RNA-activated protein kinase (PKR), which are not expressed in parental U118 cells, were constitutively expressed in IFNalpha transfectants. These results indicate a differential responsiveness among these four genes to constitutive IFNalpha expression, and suggest that the suppression of U118-transformed phenotypes by IFNalpha transfection may be mediated by the induction of specific IFN response genes thought to have a negative growth-regulatory function.

Glucosylceramide synthetase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol exhibits a novel decarcinogenic activity against Shope carcinoma cells.

The glucosylceramide synthetase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) was tested to determine whether it could exhibit anti-tumor activity against two different Shope carcinoma cell lines. The cell growth was suppressed in a dose-dependent manner in the presence of D-PDMP. This supression seem to be accounted for by prolongation of the lag phase and this phenomenon was especially marked in the undifferentiated cell line. The growth suppression was also partly explained by direct inhibition of cell proliferation because the suppression was released by removing the agent from the medium. The treated cells became morphologically differentiated with lower density at confluence and regained contact inhibition in flask culture. Colony-forming ability in soft agar, which has been reported to be closely correlated with tumorigenicity, was also inhibited dose-dependently in the presence of D-PDMP. These results suggested that D-PDMP could exhibit a novel decarcinogenic activity against Shope carcinoma cells.

Molecular cloning and characterization of a cDNA showing tumor-suppressive activity in v- src-transformed 3Y1 rat fibroblasts

Differential screening procedure was employed to isolate new genes specifically down-regulated in RSV-transformed rat 3Y1 cells (SR-3Y1) compared with parental 3Y1 cells. Among those, one clone termed N03 showed nearly complete down-regulation in v- src-, v- mos and SV40-transformed 3Y1 cells. Nucleotide sequence analysis showed that N03 protein is novel and is composed of 178 amino acid residues in length. Introduction of N03 cDNA into SR-3Y1 cells using an expression vector resulted in suppression of transformed phenotypes such as high growth rate, colony-forming ability in soft agar and tumorigenicity in nude mice.

A Novel Mutation within the Kinase Domain of v-abl Gene Responsible for Temperature-Sensitive Colony-Forming Ability in Soft Agar

Mutation sites of unique temperature-sensitive (ts) mutants of Abelson murine leukemia virus(A-MuLV) that exhibited the ts phenotype in colony-forming ability in soft agar, but not in morphological transformation, were determined. Cloning and sequencing analysis of the full viral genomes of five independent ts mutants revealed a total of 10 mutation sites: 3 mutations (bp714, 742, and 817) located in the 5′ untranslated region between LTR and gag gene; 4 mutations (bp1227, 1229, 1512, and 1634) in the gag gene; 3 mutations (point mutations at bp2764 and 3265, and 2-base deletion of bp3448 and 3449) in the abl gene. To determine the mutation sites critical for the ts phenotype in colony-forming ability in soft agar, hybrid viruses were constructed by exchanging the corresponding restriction fragments between wild-type and ts A-MuLV. The hybrid virus containing only the point mutation (T → G) at bp 2764 that exchanged leucine with arginine exhibited the ts phenotype in colony-forming ability in soft agar. Thus, a novel mutation within the kinase domain of the abl gone critical for the ts phenotype in colony-forming ability in soft agar was determined here.

Suppression of anchorage-independent growth of human glioblastoma cell by major histocompatibility complex class I gene-transfection.

The host's immune system discriminates tumor cells from normal cells by recognizing the major histocompatibility complex (MHC) class I antigen expressed on the tumor cell membrane. However, the role of MHC class I antigen in tumor cells has not yet been clarified. In this study, the influence of MHC class I antigen expression on the tumorigenicity of a human glioblastoma cell line (KMG4) is examined. Barely detectable levels of MHC class I messenger ribonucleic acid were found to express in KMG4 cells by Northern blot analysis using mouse MHC class I (H-2Ld) and human leukocyte antigen (HLA)-B7 genes as probes. The H-2Ld gene connected at the downstream end of murine mammary tumor virus (MMTV)-promoter was cotransfected with the neomycine-resistant gene pSV2-neo into KMG4 cells, and the drug-resistant cells were selected. The KMG4 cells (KMG4-MMTV-Ld), which acquired the MHC class I gene were detected by Northern blot analysis with H-2Ld as the probe, and by immunohistochemistry using the H-2Ld-specific monoclonal antibody. Tumorigenicity, as determined by colony-forming ability in soft agar, was then compared between MHC class I-expressing KMG4-MMTV-Ld and nonexpressing control cells. The MHC class I-expressing cells were found to be deprived of colony-forming ability, indicating that MHC class I antigen could negatively influence the anchorage-independent cell growth of the human glioblastoma cell line KMG4.

Effects of a synthetic polypeptide encoded by the p14–6, a cDNA clone with antioncogene activity, on malignant transformed DT cells

A synthetic polypeptide, pt27, which is encoded by a cDNA clone with antioncogene activity, p14–6, is found to be able to reduce remarkably the soft agar colony formation ability of part of DT cells and to raise their resistance to the ouabain toxicity. This shows that the pt27 peptide can affect the DT cells in a manner similar to the p14–6 clone and provides evidence that the reverting action of the p14–6 to DT cells may be exerted by the expression of its cDNA.

Tumorigenic conversion of xeroderma pigmentosum lymphoblastoid cells without karyotypic alteration.

In order to examine the process of malignant transformation of human somatic cells, we studied the tumorigenic conversion of an Epstein-Barr-virus-immortalized lymphoblastoid cell line (LCL) derived from a patient with xeroderma pigmentosum (XP) complementation group A. Repeated irradiation of the XP cells, XP7NI, with UV-light and subsequent treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the acquisition of tumorigenicity in athymic nude mice. The tumorigenicity of XP7NI cells was also induced by TPA treatment alone. The tumors formed in athymic mice were of B-cell lymphoma with characteristic histology, cell surface immunoglobulins and an antigen as detected by a B-cell-specific monoclonal antibody (MAb), CD20. The surface immunoglobulins and the HLA type of these tumor cells were identical with those of the parental cells. These malignantly transformed cells retained the same UV sensitivity, serum requirement, colony-forming ability in soft agar, and normal human karyotype as the parental cells. Unlike other tumorigenic lymphoblastoid cell lines, this XP lymphoblastoid cell line provides a unique case in that process(es) leading to tumorigenicity may be induced by UV and TPA without apparent karyotypic changes.

Transforming growth factor α

Transforming growth factor-α (TGF-α) was first detected in the culture medium of certain retrovirus-transformed cell lines as an activity which binds to the receptor for epidermal growth factor (EGF) and thereby inhibits the binding of EGF. Subsequent examination showed that this factor is made by many other transformed cell lines but not by adult normal cells in culture (Todaro et al. 1985). Addition of sarcoma growth factor, as it was first called, to rat fibroblasts of the NRK cell line reversibly induced profound morphological changes and colony-forming ability in soft agar (De Larco and Todaro 1978; Todaro et al. 1985). The capacity of this factor to confer a transformed phenotype upon NRK cells, together with the fact that it is synthesized by transformed cells, led to the name transforming growth factor.

Inconsistent effects of all-trans retinoic acid on different clones of chemically transformed rat liver epithelial cells.

The effects of all-trans retinoic acid (RA) on the growth and biochemical properties of five clonal strains of neoplastically transformed rat liver epithelial cells were studied. These cell strains were derived clonally from a single line of normal diploid rat liver epithelial cells that had been transformed by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The results show that RA induces inconsistent alterations in selected phenotypic properties of these five different cell strains. Retinoic acid either depressed, enhanced or produced no effect on the colony-forming ability in soft agar, on the activity of gamma-glutamyl transpeptidase, on the amount of cell-associated fibronectin, and on the binding capacity of 125I-epidermal growth factor (EGF). The only consistent correlation observed among cell strains was between the cellular ability to grow in soft agar and the amount of cell-associated fibronectin. Enhancement of anchorage-independent growth by retinoic acid was not mediated through changes in the number of EGF receptors. Our data demonstrate that the responses to retinoic acid of clonal subpopulations of chemically transformed rat liver epithelial cells are inconsistent, even when the clonal subpopulations are derived from a common precursor.

Two-step chromosomal control of tumorigenicity of Chinese hamster cells in nude mice.

A simple method for microinjecting isolated chromosomes into a single living cell under an inverted microscope has been developed. Of the 368 injected cells, 85 were able to form a colony and could be cloned. Clones of Chinese hamster V79 cells microinjected with chromosomes isolated from murine D56 cells [V79 (D56) cells] were tested for tumorigenicity in immunodeficient nude mice and for colony-forming ability in soft agar. Untreated recipient V79 cells were highly tumorigenic and had a high colony-forming ability in soft agar. In contrast, two out of the 21 microinjected clones tested were non-tumorigenic in nude mice and had only weak colony-forming ability in soft agar. The chromosome banding pattern was analyzed in microinjected clones and tumors derived from cells of these clones. In cells of the two non-tumorigenic clones, a telocentric chhromosome 1 (t1) was specifically involved in translocations with other chromosomes or chromosome fragments. In all tumor cells obtained from nude mice, a supernumerary piece or a whole biarmed chromosome 14 (b14) was specifically found. The results suggest that the t1 chromosome bears the gene which controls in vitro transformation and that the additional genetic change, i.e. the extra piece of b14 chromosome, was required for tumor formation in vivo.

Irreversible loss of negative surface charge and loss of colony-forming ability in Burkitt lymphoma cells after X-irradiation

The electrokinetic behavior of Burkitt lymphoma P 3H-R1 cells were studied following X-irradiation. The surface charge of irradiated cells decreased progressively with time after X-ray treatment, as compared with a mean mobility value (−0.677 ± 0.045 μsec −1 V −1 cm) of non-irradiated cells. The frequency distribution of cells treated with 3 000 R 4 h previously showed quite a different peak of lower mobility than that of non-irradiated cells with the boundary of −0.550 μsec −1 V −1 cm. Percentages of cells which showed the higher mobility than the boundary value 4 h after irradiation with 30, 50, 65, 100, 200 or 3 000 R were in accord with the percentage of cells which showed colony-forming ability in soft agar, respectively. Following cell proliferation the population of electrokinetically intact cells increased with time after irradiation; these values also coincided with the population of colony-forming cells. Thus, the present results indicate that there is a close relation between loss of negative surface charge and loss of colony forming ability in irradiated cells, suggesting that loss of colony forming ability of the cells may be determined as early as 4 h after irradiation.