Gertrude Case Buehring

Elizabeth A. Eby

Cell Line Cross-contamination

HeLa was the first human cell line established (1952), and became one of the most frequently used lines due to its hardiness and rapid growth rate. During the next two decades, the development of other human cell lines mushroomed. One reason for this became apparent during the 1970s, when it was demonstrated that many of these cell lines had been overgrown and replaced by fast growing HeLa cells inadvertently introduced into the original cultures. Although the discovery of these “HeLa contaminants” prompted immediate alarm, how aware are cell culturists today of the threat of cell line cross-contamination? To answer this question, we performed a literature search, and conducted a survey of 483 mammalian cell culturists to determine how many were using HeLa contaminants without awareness of their true identity, and how many were not using available means to insure correct identity. Survey respondents included scientists, staff, and graduate students in 48 countries. HeLa cells were used by 32% and HeLa contaminants by 9% of survey respondents. Most were also using other cell lines, yet only about a third of respondents were testing their lines for cell identity. Of all the cell lines used, 35% had been obtained from another laboratory instead of from a repository, thus increasing the risk of false identity. Over 220 publications were found in the PubMed database (1969-2004) in which HeLa contaminants were used as a model for the tissue type of the original cell line. Overall, the results of this study indicate a lack of vigilance in cell acquisition and identity testing. Some researchers are still using HeLa contaminants without apparent awareness of their true identity. The consequences of cell line cross-contamination can be spurious scientific conclusions; its prevention can save time, resources, and scientific reputations. Gertrude Case Buehring, Elizabeth A. Eby and Michael J. Eby. Cell Line Cross-contamination: How Are Mammalian Cell Culturists of the Problem and How To Monitor It?, In Vitro Cellular and Developmental Biology – Animal, 40:211-215, 2004.


SOMATIC EMBRYOGENESIS IN CARROT SUSPENSION CULTURES

 

Various aspects of somatic embryogenesis in carrot suspension cultures were reviewed on the basis of results obtained in our laboratory. We have established high-frequency and synchronous somatic embryogenesis systems needed for biochemical and molecular analysis. Using these systems, four phases of somatic embryogenesis were identified. The importance of expression of polarities in these phases, particularly from single cells to embryogenic cell clusters, in determining somatic embryogenesis, is emphasized. At the molecular level, genes expressed during somatic embryogenesis were described, and they were classified into three categories: (1) genes involved in cell division, (2) genes involved in organ formation, and (3) genes specific for the process of somatic embryogenesis. From the results obtained, it is concluded that discrete developmental phases in carrot somatic embryogenesis are characterized by distinct biochemical and molecular events, but much remains to be understood. Atsushi Komamine, N. Murata, and K. Nomura, 2004 SIVB Congress Symposium Proceeding: Mechanisms of Somatic Embryogenesis in Carrot Suspension Cultures: Morphology, Physiology, Biochemistry and Molecular Biology, In Vitro Cellular and Developmental Biology – Plant, 41:6-10, 2005.



Pamela J. Weathers, G. Bunk and M. C. McCoy

Artemisia annua Hairy Roots
Few studies have focused on the effect of a broad range of phytohormones on growth and secondary metabolism of single hairy root species. We measured growth, development, and production of the antimalarial drug, artemisinin, in Artemisia annua hairy roots in response to the five main hormones: auxins, cytokinins, ethylene, gibberellins, and abscisic acid. Single roots grown in six-well plates in medium B5 with 0.01 mg/l (0.029 µM) GA3 produced the highest values overall in terms of the number of lateral roots, length of the primary root, lateral root tip density, total lateral root length, and total root length. When the total root lengths are compared, the best conditions for stimulating elongation appear to be: GA 0.01 mg/l (0.029 µM) > ABA 1.0 mg/l (3.78 µM) = GA 0.02 mg/l (0.058 µM). Bulk yields of biomass were inversely proportional to the concentration of each hormone tested. All cultures provided with ABA yielded the highest amount of biomass. Both 6-benzylaminopurine and 2-isopentenyladenine (2iP) inhibited root growth, however, only 2iP stimulated artemisinin production, more than twice that of the B5 controls, and more than any other hormone studied. These results will prove useful in increasing hairy root growth and artemisinin production. Pamela J. Weathers, G. Bunk and M. C. McCoy. The Effect of Pytohormones on Growth and Artemisinin Production in Artemisia annua Hairy Root, In Vitro Cellular and Developmental Biology – Plant, 41:47-53, 2005.


Andrew Yen

David M. Lin

HL-60 Leukemic Cell Differentiation

Retinoic acid, bromodeoxyuridine, and the D205 mutant polyoma middle T antigen affect the expression of a common ensemble of proteins in HL-60 human myeloblastic leukemia cells. Each of these agents is known to be able to prime HL-60 cells and accelerate subsequently induced myeloid or monocytic differentiation and G0 cell cycle arrest, suggesting that they have equal or identical cellular targets relevant to the early stages of inducing cell differentiation and G0 arrest. As a test of this possibility, a survey of protein expression changes induced by RA, BrdU or D205 transfection was performed. RA induced numerous changes within hours. BrdU caused larger numbers of changes, whereas D205 caused a more limited number. Among the hundreds of affected proteins detected, there were comparable numbers of up or down regulated proteins. A small number changed between undetectable and detectable expression. The affected proteins were not restricted to a single functional class and included transcription factors, receptors, signaling molecules, cytoskeletal molecules, and effectors of various cellular processes such as DNA replication, transcription, and translation. The intersect of the sets of proteins affected by RA, BrdU and D205 was identified to determine if these agents regulated a common subset of proteins. This ensemble contained the commonly up regulated proteins: AF6, ABP-280, ENC-1, ESE 1, MAP2B, NTF2, Casein Kinase, IRF1, SRPK2, Rb 2, RhoGDI, P47phox, CD45, PKR, and SIIIp15. The commonly down regulated proteins were: SHC, Katanin, Flotillin-2/ESA, EB 1, p43/EMAPIIprecursor, Jab1, FNK. The composition of the ensemble suggested three apparent themes for cellular processes that were affected early. The themes reflected the ultimate fate of the treated precursor cells as a mature myeloid cell, namely a cell whose hallmarks are 1) motility to migrate to a target and phagocytize it, 2) inducible oxidative metabolism to reduce the target with superoxide from a respiratory burst, and 3) biosynthetic slow down consistent with conversion from cell proliferation to quiescence. Interestingly RA appears to induce aspects of an interferon-like response of potential significance as part of a biosynthetic slow down leading to cell cycle arrest. In conclusion, 3 biologically disparate ways to prime cells to differentiate were used to filter out a small ensemble of commonly regulated proteins which group as either microtubule associated, oxidative metabolism machinery, or effectors of cellular responses to interferon. Andrew Yen, David M. Lin, Thomas J. Lamkin and Susi Varvayanis. Retinoic Acid, Bromodeoxyuridine, and the ?205 Mutant Polyoma Virus Middle T Antigen Regulate Expression Levels of a Common Ensemble of Proteins Associated with Early Stages of Inducing HL-60 Leukemic Cell Differentiation, In Vitro Cellular and Developmental Biology – Animal, 40:216-242, 2004.


Arabidopsis Cell Fate Specification

Root epidermis development in Arabidopsis provides a simple and powerful model for studying cell fate specification. Cellular, molecular, and genetic approaches have been used to define many genes, and their corresponding proteins, that are essential for the position-dependent specification of the two-root epidermal cell types. These studies have led to a working model in which a network of transcriptional regulators that is influenced by positional cues establishes differences in gene expression in neighboring cells through a set of positive and negative feedback loops. The continued analysis of this experimental system is likely to provide new insights into mechanisms of transcriptional regulation and cell-cell interactions during development. J. Schiefelbein, C. Bernhardt, S. H. Kwak, and M. Simon. 2004 SIVB Congress Symposium Proceeding: Cell Fate Specification During Development of the Arabidopsis Root Epidermis, In Vitro Cellular and Developmental Biology – Plant, 41:1-5, 2005.