Transformation Of Cells In Culture Research Articles

The small GTP-binding protein rac is a regulator of cell death in plants.

Cell death plays important roles in the development and defense of plants as in other multicellular organisms. Rapid production of reactive oxygen species often is associated with plant defense against pathogens, but their molecular mechanisms are not known. We introduced the constitutively active and the dominant negative forms of the small GTP-binding protein OsRac1, a rice homolog of human Rac, into the wild type and a lesion mimic mutant of rice and analyzed H(2)O(2) production and cell death in transformed cell cultures and plants. The results indicate that Rac is a regulator of reactive oxygen species production as well as cell death in rice.

Mutagensis and cell transformation in cell culture.

The current lack of continuous prawn cell lines suitable for the isolation and growth of prawn viruses is a major setback for diagnosis of viral diseases of prawns; isolation and identification of the causative agents are severely hindered and the development of other diagnostic procedures is slowed. To date, there are few, if any, examples of continuous prawn cell lines suitable for virus isolation and growth. Recent studies indicate that clastogenic agents such as ionising radiation are more effective than powerful point mutagens as immortalizing agents for mammalian cells. Such studies with mammalian cell cultures indicate that certain mutagenic agents and/or prolonged treatment with a combination of mutagens may prove more useful in the production of immortal prawn cell lines in vitro rather than other, more limited and specific procedures. This report provides a brief review of the use of mutagens to induce immortalisation -- the acquisition of unlimited growth potential -- in mammalian cells in culture, i.e. the production of continuous cell lines from primary cultures. Aspects of cell transformation vs. immortalisation, the control of cell growth in vitro, and the application of mutagenic agents to induce immortalisation in primary cultures of prawn tissues will be discussed.

Cooperativity between the polyamine pathway and HER-2neu in transformation of human mammary epithelial cells in culture: role of the MAPK pathway.

Our experiments were designed to test the cooperativity between the polyamine pathway and HER-2neu in inducing transformation of human mammary epithelial cells in culture. Using the MCF-10A breast epithelial cell line, we observed that induction of overexpression of ornithine decarboxylase (ODC) (the first rate-limiting enzyme in polyamine biosynthesis) markedly potentiated the anchorage-independent growth stimulating effect of the beta2 isoform of neu differentiating factor (NDF) known to activate HER-2neu in MCF-10A cells. ODC overexpression, on the other hand, did not enhance growth in liquid culture, thus pointing to a specific effect on transformation rather than proliferation. ODC-overexpressing MCF-10A cells exhibited increased MAPK phosphorylation in response to administration of NDF and/or epidermal growth factor (EGF). In contrast, the phosphorylation of the members of the stress-activated protein kinase cascade p38 and SEK were not affected by ODC overexpression. Of note, in the absence of EGF and NDF, ODC overexpression failed to induce both clonogenicity and MAPK activation. These results suggest that increased polyamine biosynthetic activity critically interacts with HER-2neu in promoting human mammary cell transformation in culture and that the MAPK cascade is an important mediator of this interaction. If confirmed in future in vivo studies, our results may identify important new targets for the chemoprevention of human breast cancer.

Cooperativity between the polyamine pathway and HER‐2neu in transformation of human mammary epithelial cells in culture: Role of the MAPK pathway

Our experiments were designed to test the cooperativity between the polyamine pathway and HER-2neu in inducing transformation of human mammary epithelial cells in culture. Using the MCF-10A breast epithelial cell line, we observed that induction of overexpression of ornithine decarboxylase (ODC) (the first rate-limiting enzyme in polyamine biosynthesis) markedly potentiated the anchorage-independent growth stimulating effect of the β2 isoform of neu differentiating factor (NDF) known to activate HER-2neu in MCF-10A cells. ODC overexpression, on the other hand, did not enhance growth in liquid culture, thus pointing to a specific effect on transformation rather than proliferation. ODC-overexpressing MCF-10A cells exhibited increased MAPK phosphorylation in response to administration of NDF and/or epidermal growth factor (EGF). In contrast, the phosphorylation of the members of the stress-activated protein kinase cascade p38 and SEK were not affected by ODC overexpression. Of note, in the absence of EGF and NDF, ODC overexpression failed to induce both clonogenicity and MAPK activation. These results suggest that increased polyamine biosynthetic activity critically interacts with HER-2neu in promoting human mammary cell transformation in culture and that the MAPK cascade is an important mediator of this interaction. If confirmed in future in vivo<0R> studies, our results may identify important new targets for the chemoprevention of human breast cancer. Int. J. Cancer 76:563–570, 1998.© 1998 Wiley-Liss, Inc.

STP and Tip are essential for herpesvirus saimiri oncogenicity.

Mutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS deltaSTP-C488 and HVS deltaTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.

Modulation of glutamate neurotoxicity in the transformed cell culture by monoamine oxidase inhibitors, clorgyline and deprenyl.

Addition of 30mM glutamate to the culture medium decreased growth of rat glioma C6 cells accompanied by a decrease of DNA synthesis and an increase of lactate dehydrogenase (LDH) detected in the conditioned medium. The presence of 1 microM deprenyl attenuated the glutamate effect on cell growth only during the first 24-48 h incubation and had a minor influence on the glutamate-induced decrease of DNA synthesis. Clorgyline (1 microM) potentiated glutamate-induced DNA synthesis during the first 24 h incubation without significant influence on the cell growth. Deprenyl slightly attenuated the glutamate-induced LDH increase during 24 h incubation but potentiated the glutamate effect at 96 h. Clorgyline decreased the glutamate influence at 24 h and especially 96 h. All these effects were observed in the absence of exogenous monoamines in the culture medium. These results suggest that in transformed cells monoamine oxidase (MAO) inhibitors may influence processes of cell death via MAO-independent mechanisms.

Use of plant cell cultures in biotechnology

Plant cell cultures are being widely used in scientific studies on the physiology, biochemistry and molecular biology of primary and secondary metabolism, developmental regulation and cellular responses to pathogens and stress. In this chapter the significance of plant cell cultures in biotechnology is discussed with special emphasis on commercial production of secondary metabolites and pharmaceuticals, the potential of genetically transformed cell cultures, photosynthetically active cell cultures, production of somatic embryos, and novel assay systems based on the use of plant cells. Future aspects of biotechnical applications with respect to the potentials and limitations of these approaches are assessed, particularly in comparison with the productivity of lower eucaryotes.

Tanshinone production in Ti-transformed Salvia miltiorrhiza cell suspension cultures

Transformed cell cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium tumefaciens strain C58. The transformed cells in suspension formed macroscopic clumps of cell aggregates up to 2–3 cm in size rather than homogeneous cell suspensions. These transformed cells grew well in hormone-free media. It was found that the B5 medium supported the best growth while the 6,7-V medium promoted tanshinone production in the transformed cell suspension cultures. The effect of initial sucrose concentrations on cell growth was also studied. The best growth was observed when cells were cultivated in the B5 medium containing 30 g l−1 sucrose. Although low levels of tanshinones were produced in fast growing cell cultures, there existed a rapid increase in tanshinone production when the cell aggregates were transferred to the fresh yeast-extract-containing medium. By this two-stage culture method, about 22 mg tanshinones were produced in 1 liter of medium. Green cell aggregates were formed when cells were cultured under illumination. Light was found to have an inhibitory effect on tanshinone biosynthesis. A sensitive high-performance liquid chromatographic method was developed for the measurement of tanshinones. Cryptotanshinone, tanshinone I and tanshinone IIA were identified from the transformed cultures.

Transformation and tumorigenic properties of a mutant polyomavirus containing a middle T antigen defective in Shc binding.

Polyomavirus middle T antigen is phosphorylated on several tyrosine residues which act as binding sites for cellular proteins, including phosphatidylinositol 3-kinase, Shc, and phospholipase C-gamma. In this report we describe the transforming properties and tumor-inducing ability of a polyomavirus that contains a single-site mutation in middle T antigen which changes a tyrosine residue at amino acid position 250 to serine. This mutation disrupts the association of middle T with the transforming protein Shc. The mutant virus is weakly transforming, inducing foci which are smaller and of different morphology than those of the wild type. Although the virus induced tumors in close to 100% of inoculated mice, the spectrum of tumors and their morphology were altered compared to those of wild-type virus. The mutant virus induced a reduced frequency of kidney and thymic tumors. Both the mammary gland and the thymic tumors that were induced were histologically distinct from those induced by wild-type polyomavirus. These results demonstrate that the signal transduction pathway that is deregulated by the middle T-Shc association is important for full transformation of cells in culture and for tumor induction in some target tissues in the mouse-polyomavirus system.

Tumor induction by a transformation-defective polyoma virus mutant blocked in signaling through Shc.

Transformation of cells in culture by polyoma virus requires integration of signals downstream of middle T-Shc and middle T-phosphatidylinositol 3-kinase interactions, but the same is not true for induction of tumors in the mouse. Thus, a middle T mutant defective in transformation and blocked in binding Shc is able to induce a broad spectrum of tumors after inoculation into newborn mice. The "tumor profile" induced by the mutant shows enhancement of tumors at some sites and reductions at others but otherwise resembles that induced by the wild-type virus. A nontransforming double-mutant blocked in binding phosphatidylinositol 3-kinase as well as Shc is severely affected but still induces some tumors. These results show that pathways that must cooperate to induce full transformation of cells in vitro can act independently and are to a large extent redundant in tumor induction.

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A.

A yeast two-hybrid screen has identified HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1--a high affinity binding site, mediated by an LXCXE motif and a separate low affinity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N- and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP co-activator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture.

Human prostate carcinogenesis.

Prostate cancer is a major medical problem that is expected to affect over 300,000 American men and cause over 40,000 deaths in 1997. Despite its widespread prevalence and because of the difficulties in clinical diagnosis and treatment of the disease, the etiological mechanism underlying prostate carcinogenesis remains poorly understood. Elucidation of the mechanism of prostate tumorigenesis has been slowed by a lack of tumor tissues and the limited number of human cell lines available for study. In vitro human cell models to study the molecular biology of prostate cancer progression are urgently needed. Normal human prostate cells require immortalization to provide a practical system for transformation studies. Neoplastic transformation of human prostate epithelial cells in culture has been achieved recently in a stepwise fashion--immortalization of primary cells in culture and conversion of the immortalized cells to a tumorigenic state. Reviewed here are the steps involved in the neoplastic transformation of human prostate cells. To provide an insight into the molecular and genetic mechanisms involved in the conversion of normal cells to a neoplastic state of growth, the authors have attempted to put into perspective the history of human prostate epithelial cell transformation by a combination of carcinogenic agents, and to discuss the current state-of-the-art in transformation of human prostate epithelial cells in culture.

Functional interaction between the SH2 domain of Fyn and tyrosine 324 of hamster polyomavirus middle-T antigen

Middle-T antigen of mouse polyomavirus (MomT) associates with the cellular tyrosine kinases c-Src, c-Yes, and Fyn, while middle-T antigen of hamster polyomavirus (HamT) exclusively binds Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. Here we show that the kinase domain of Fyn is sufficient for association with MomT but not for binding of HamT. We further demonstrate that a Fyn mutant lacking the SH2 domain is able to bind MomT but fails to associate with HamT, indicating that the SH2 domain of Fyn is essential for stable association with HamT. HamT, but not MomT, contains a tyrosine residue, Tyr-324, in the sequence context YEEI. Mutation of Tyr-324 to phenylalanine led to a drastic reduction of associated Fyn and abolished the oncogenicity of HamT. This suggests that Tyr-324 is the major phosphotyrosine residue mediating the binding of HamT to the SH2 domain of Fyn. These findings show that mouse and hamster polyomaviruses use different strategies to target Src-related tyrosine kinases.

Characterization of Appican, the Chondroitin Sulfate Proteoglycan Form of the Alzheimer Amyloid Precursor Protein

In this report we focus on the characterization of appican, the chondroitin sulfate proteoglycan form of amyloid precursor protein (APP), and the role that it and other proteoglycans may play in AD. Appican is expressed by certain transformed cell lines of neural origin, namely C6 cells and N2a neuroblastomas. It is detected in both human and rat brain and in primary cultures is expressed by astrocytes, but not neurons. The core protein of appican has been shown to be an alternatively spliced isoform of APP, lacking exon 15 of the APP gene, originally identified in leukocytes (L-APP). Splicing out of exon 15 results in the joining of exons 14 and 16, and formation of an Asp-Xaa-Ser-Gly consensus sequence for chondroitin sulfate chain attachment to serine 619 of L-APP, which lies 16 amino acids upstream of the A β peptide sequence. Mutation of this serine residue to an alanine prevented chondroitin sulfate chain addition to the core protein. Levels of appican expression could be regulated by growth conditions independently of APP, suggesting that these molecules may serve distinct physiological roles within the cell. Morphological changes were also observed in both astrocytic and transformed cell cultures, that appeared to reflect changes in levels of appican expression. Preliminary data suggest that appican may be a strong cell adhesion molecule. Transfected C6 glioma cells overexpressing appican remained attached to tissue culture dishes markedly better than either C6 cells over-expressing exon-15 containing APP or WT C6 cells. Appican-enriched extracellular matrix (ECM) was also observed to serve as a much better substrate for attachment of N2a neuroblastomas, pheocromocytoma PC12 cells and primary astrocytes compared to APP enriched ECM.

Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases.

Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino-terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy-terminal regulatory tyrosine and the SH2 domain prevents association with middle-T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src.

Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation.

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.

Shikonin production by extractive cultivation in transformed-suspension and hairy root cultures of Lithospermum erythrorhizon.

Effects of in situ extraction, fungal elicitation, a permeabilizing agent, and the oxygen transfer rate on shikonin production in transformed suspension and hairy root cultures of Lithospermum erythrorhizon were studied. Shikonin production with in situ extraction in transformed cell and hairy root cultures by n-hexadecane was 7.6 and 3 times higher than those of the control culture. Shikonin production of transformed L. erythrohizon increased with the enhanced gas exchange, and in situ extraction also increased sucrose consumption and shikonin production. The optimal volume of n-hexadecane in the hairy root culture was similar to that in the transformed cell cultures. In situ extraction at an earlier stage significantly enhanced shikonin production both in transformed cell and hairy root cultures. Dimethylsulfoxide used as a permeabilizing agent was harmful to cell growth and shikonin production, and permeabilizing was unnecessary when in situ extraction was applied. This occurred because with the solvent addition, most shikonin was spontaneously released from the cells and was dissolved in the solvent layer. The combined addition of n-hexadecane of the extract of the fungus Penicillium as an elicitor seemed to result in a higher production of shikonin both in cell suspensions and transformed root cultures. However, an increase of shikonin induction by fungal elicitation in a hairy root culture was not significant in comparison with that of normal cell cultures.

Protein farnesyltransferase inhibitors block the growth of ras-dependent tumors in nude mice.

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its transforming activity. Cell-active inhibitors of the enzyme that catalyzes this reaction, protein farnesyltransferase, have been shown to selectively block ras-dependent transformation of cells in culture. Here we describe the protein farnesyltransferase inhibitor 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl] pentyloxy-3-phenylpropionylmethioninesulfone methyl ester (L-739,749), which suppressed the anchorage-independent growth of Rat1 cells transformed with viral H-ras and the human pancreatic adenocarcinoma cell line PSN-1, which harbors altered K-ras, myc, and p53 genes. This compound also suppressed the growth of tumors arising from ras-transformed Rat1 cells in nude mice by 66%. Under the same conditions, doxorubicin inhibited tumor growth by 33%. Control tumors formed by v-raf- or v-mos-transformed Rat1 cells were unaffected by L-739,749. Furthermore, mice treated with L-739,749 exhibited no evidence of systemic toxicity. This is a demonstration of antitumor activity in vivo using a synthetic small molecule inhibitor of protein farnesyltransferase.

Analysis of Epstein-Barr virus in Hodgkin's disease: experience of a single university hospital in Korea.

Hodgkin's disease is known to be associated with Epstein-Barr virus (EBV) infection in Western countries, and viral nucleic acids and proteins have been identified within Reed-Sternberg (RS) cells, which are the histopathologic hallmark of the disease process. Twenty-five cases of Hodgkin's disease from a single university hospital in Korea were studied for evidence of EBV by in situ hybridization for EBV DNA and RNA and immunohistochemistry for an EBV latent protein. EBV nucleic acids were studied by a rapid (60 minutes) in situ hybridization procedure, which utilized biotinylated DNA probes specific for the following nucleic acid sequences: (1) EBV EBER1 RNA (an abundant RNA sequence expressed during latent EBV infection), (2) EBV NotI repeats (a tandemly repeated DNA sequence, which has been established to identify amplified EBV genome in lytic EBV infection), and (3) BAM HI W (a DNA sequence reiterated 11 times within the viral genome). In addition, immunohistochemistry for EBV latent membrane protein, a protein that is capable of inducing cellular transformation in cell culture, was also performed. EBV was identified within the neoplastic RS cells by at least one method in 19/25 cases (76%). The mixed cellularity subtype was the most common subtype associated with EBV infection (11/13-85%). In situ hybridization for EBV EBER1 RNA was the most sensitive method for EBV detection and was present in 17/25 cases. A significant proportion of Korean Hodgkin's disease cases is associated with EBV infection.

Viruses and human cancer.

Epstein-Barr virus (EBV), hepatitis B virus (HBV), HTLV-I and some human papillomaviruses (HPVs) appear to contribute to the development of a large proportion of certain human cancers. Although the epidemiological evidence linking infection with these viruses to malignancies is generally convincing, only for the HPVs have experimental systems indicated a clear role for some of the HPV encoded genes in tumour cell growth. In these cases, molecular analysis is revealing the mechanisms by which the virally encoded oncogenes function to perturb the normal regulation of cell growth. For HTLV-I, EBV and HBV the mechanism by which the viruses contribute to tumour cell growth is obscure, even though much has been learned about cell transformation in culture by these viruses. A detailed understanding of the mechanism of oncogenesis will be required to design therapeutic drugs for the treatment of these cancers. Prophylactic vaccination resulting in prevention of infection may be an effective approach to reduce the incidence of some of these common cancers.