Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions

Harasawa, Ryô; Mizusawa, Hiroshi; Nozawa, Kohei; Nakagawa, Tomoko; Asada, Kiyozo; Kato, Ikunoshin

doi:10.1016/0923-2508(93)90057-9

Cited by 89 publications

(63 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, it has been reported that M. hyorhinis infection activates nNOS expression in human neuroblastoma cell line YT-nu [9]. Therefore, it is necessary to ascertain the absence of mycoplasma in cell cultures prior to study [11].…”

Section: Discussionmentioning

confidence: 99%

Nitric Oxide Causes Anoikis through Attenuation of E-Cadherin and Activation of Caspase-3 in Human Gastric Carcinoma AZ-521 Cells Infected with Mycoplasma hyorhinis

Obara

Harasawa

2010

The Journal of Veterinary Medical Science

View full text Add to dashboard Cite

ABSTRACT. Mycoplasma hyorhinis (M. hyorhinis) infection leads cultured cells to various biological alterations in cell metabolism including apoptosis. Apoptosis induced by M. hyorhinis has mainly been considered to be due to mycoplasmal endonucleases. We previously reported that apoptosis in a human carcinoma cell line AZ-521 infected with M. hyorhinis was enhanced by addition of L-ascorbic acid to cell cultures. Since both L-ascorbic acid addition and M. hyorhinis infection activated cellular iNOS, we examined the hypothesis that nitric oxide (NO) exerts an apoptotic effect on M. hyorhinis-infected cells and down-regulates E-cadherin. In this study, we showed that M. hyorhinis infection activates iNOS mRNA synthesis, NO production, and caspase-3 activity and attenuates E-cadherin mRNA synthesis by quantitative real-time PCR, Griess assay and fluorescence caspase-3 detection. L-NAME decreased the numbers of apoptotic cells through inhibition caspase-3 activity. Our results indicate that NO causes anoikis throughout attenuation of E-cadherin and activation of caspase-3 in human gastric carcinoma cell line AZ-521 cells infected with M. hyorhinis.KEY WORDS: anoikis, apoptosis, E-cadherin, Mycopalsma hyorhinis, nitric oxide.J. Vet. Med. Sci. 72 (7): [869][870][871][872][873][874] 2010 Mycoplasmas are the smallest self-replicating prokaryotes, lack rigid cell walls and belong to the class Mollicutes [25]. Most mycoplasma species inhabit plants, insects, animals and humans as normal flora in their hosts. Although some are pathogenic, many possess an opportunistic character [22].Mycoplsma hyorhinis (M. hyorhinis) causes polyserositis [7], otitis media [19], arthritis [12] and pneumonia [1] in piglets. These mycoplasmal infections are responsible for economical losses on swine farms. M. hyorhinis is also a major and serious contaminant in cell cultures [2]. In addition, it has been demonstrated in human gastric carcinoma tissues [26], and it has been suggested that lipoprotein P37 of M. hyorhinis contributes to invasiveness and metastasis of the tumor cells [5,14].M. hyorhinis infection has a significant impact on the physiology of cell cultures, including apoptosis, which has previously been explained by mycoplasmal endonucleases [23], production of nitric oxide (NO) by activating cellular inducible NO synthase (iNOS) and production of reactive oxygen species (ROS) in various cell lines [13]. NO is a multifunctional molecule involved in a variety of physiological and pathological processes [17]. While low concentrations of NO can protect cells from apoptosis, excess NO promotes cell death in various cell types [3].Apoptosis is characterized morphologically by cell shrinkage and chromatin condensation and biochemically by DNA laddering [30]. Detection of caspase activity is a useful assay for apoptosis. The caspase family participates in a series of reactions that are trigged in proapototic signals and result in the cleavage of substrates, and caspases are synthesized as inactive precursors that undergo proteolytic m...

show abstract

Section: Discussionmentioning

confidence: 99%

Nitric Oxide Causes Anoikis through Attenuation of E-Cadherin and Activation of Caspase-3 in Human Gastric Carcinoma AZ-521 Cells Infected with Mycoplasma hyorhinis

Obara

Harasawa

2010

The Journal of Veterinary Medical Science

View full text Add to dashboard Cite

show abstract

“…The construction and application of probes for the detection of cell culture Mycoplasmas have been reported by several groups. [18][19][20] PCRbased methods have also been developed. 3,16 However, most of the methods described earlier might be time-consuming, lack of sensitivity and might not detect all the Mycoplasma species involved in cell culture and virus stock contaminants.…”

Section: Discussionmentioning

confidence: 99%

Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

Gopalkrishna

Verma

Kumbhar

et al. 2007

Indian J Med Microbiol

View full text Add to dashboard Cite

“…Normal human mesothelial cells were cultured from ascites fluid from patients without infection or malignancy according to a protocol approved by the UCSF Committee on Human Research. All cells were confirmed to be negative for mycoplasma every 2 months by PCR analysis as previously described (26).…”

Section: Cell Culturesmentioning

confidence: 99%

Mammalian Target of Rapamycin Contributes to the Acquired Apoptotic Resistance of Human Mesothelioma Multicellular Spheroids

Barbone

Yang

Morgan

et al. 2008

Journal of Biological Chemistry

137

121

View full text Add to dashboard Cite

When grown as three-dimensional structures, tumor cells can acquire an additional multicellular resistance to apoptosis that may mimic the chemoresistance found in solid tumors. We developed a multicellular spheroid model of malignant mesothelioma to investigate molecular mechanisms of acquired apoptotic resistance. We found that mesothelioma cell lines, when grown as multicellular spheroids, acquired resistance to a variety of apoptotic stimuli, including combinations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), ribotoxic stressors, histone deacetylase, and proteasome inhibitors, that were highly effective against mesothelioma cells when grown as monolayers. Inhibitors of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway, particularly rapamycin, blocked much of the acquired resistance of the spheroids, suggesting a key role for mTOR. Knockdown by small interference RNA of S6K, a major downstream target of mTOR, reproduced the effect of rapamycin, thereby confirming the role of mTOR and of S6K in the acquired resistance of threedimensional spheroids. Rapamycin or S6K knockdown increased TRAIL-induced caspase-8 cleavage in spheroids, suggesting initially that mTOR inhibited apoptosis by actions at the death receptor pathway; however, isolation of the apoptotic pathways by means of Bid knockdown ablated this effect showing that mTOR actually controls a step distal to Bid, probably at the level of the mitochondria. In sum, mTOR and S6K contribute to the apoptotic resistance of mesothelioma cells in three-dimensional, not in twodimensional, cultures. The three-dimensional model may reflect a more clinically relevant in vitro setting in which mTOR exhibits anti-apoptotic properties.Resistance to apoptosis is a distinguishing characteristic of all tumors (1) and underlies their resistance to therapy (2). Understanding the molecular foundations of apoptotic resistance could lead to improvements in the utility of current therapies or provide new therapies altogether. In vitro, apoptotic resistance of tumor cells is usually studied using two-dimensional monolayers. However, cells can attain increased resistance when grown in three-dimensional structures, a phenomenon referred as the acquisition of multicellular resistance (3). This acquired property of tumor cells may help explain why promising findings from in vitro studies have not been easily translated into therapy (4). Consequently, in the last decade, multicellular spheroids have become a valuable tool in the study of solid tumors by representing a threedimensional system of intermediate cellular complexity between monolayer cell cultures and tumors in vivo (5, 6).Human malignant pleural mesothelioma, an aggressive thoracic cancer that arises from the pleural mesothelium, is characterized by a profound resistance to standard anti-neoplastic therapies. At present, no curative therapy is available (7). To investigate the apoptotic resistance of mesothelioma, we are increasingly utilizing in vitro three-dimension...

show abstract

Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions

Cited by 89 publications

References 9 publications

Nitric Oxide Causes Anoikis through Attenuation of E-Cadherin and Activation of Caspase-3 in Human Gastric Carcinoma AZ-521 Cells Infected with Mycoplasma hyorhinis

Nitric Oxide Causes Anoikis through Attenuation of E-Cadherin and Activation of Caspase-3 in Human Gastric Carcinoma AZ-521 Cells Infected with Mycoplasma hyorhinis

Detection of <i>Mycoplasma </i>species in cell culture by PCR and RFLP based method: Effect of BM-cyclin to cure infections

Mammalian Target of Rapamycin Contributes to the Acquired Apoptotic Resistance of Human Mesothelioma Multicellular Spheroids

Contact Info

Product

Resources

About