Alteration of gene expression profiles during mycoplasma-induced malignant cell transformation

Mycoplasmas are a group of the smallest free-living organisms that commonly infect or colonize the oral-respiratory and urogenital tracts of humans and animals. These wall-free mycoplasmas are among the few prokaryotes that can grow in close interaction with mammalian cells, often silently, for a long period of time [1]. Our previous studies have revealed that chronic and persistent infection with these seemingly low-virulence mycoplasmas could gradually but significantly affect many important biological characteristics of mammalian cells and even lead to malignant transformation [2–4].

C3H cells (C3H10T1/2), a murine embryonic cell line, rarely undergo spontaneous transformation, and are one of the few standard test systems for studying potential carcinogenic agents in animals or humans [5]. M. fermentans and M. penetrans are the most common mycoplasmas associated with HIV infection or AIDS, and have been proposed to play a co-factor role in the disease process [6]. Our earlier study revealed that chronic infections with M. fermentans and M. penetrans induced malignant transformation of C3H cells [3]. However, as opposed to earlier reports of mycoplasma-associated malignant cell transformation [7, 8], our study showed that infection by M. fermentans or M. penetrans did not lead to rapid transformation of C3H cells. Instead, the mycoplasma-mediated malignant cell transformation required a prolonged period of infection with these organisms (a long latency) and was evidently a multistage process. Although C3H cells showed prominent morphological transformation after 7 weeks of mycoplasmal infections, malignant properties associated with cell transformation could be rapidly reversed by eradicating the mycoplasma from culture with antibiotics up to 11 weeks of mycoplasmal infection (reversible stage). However, further persistent infection beyond 18 weeks resulted in an irreversible form of transformation that included prominent chromosomal alteration, the ability to form tumors in animals and high efficiency of colony formation in soft agar. At this irreversible stage, the transformed cells showed unique karyotypic changes and growth characteristics. The continued presence of the transforming mycoplasmas was no longer required for maintaining the transforming traits of the cells [3].

The multistage transformation effect associated with chronic M. fermentans and M. penetrans infections was similarly observed in our recent studies using 32D cells, a mouse hematopoietic cell line strictly dependent on IL-3 for their growth [2, 9]. Infection with M. fermentans or M. penetrans prevented apoptosis and effectively induced IL-3 independent growth of the cells. Prolonged infections with these mycoplasmas eventually lead to autonomous growth of the cells in vitro and to form tumors in mice in the absence of either IL-3 or the transforming mycoplasmas [2, 10]. Some strains of M. fermentans were very effective in immortalization of human peripheral blood mononuclear cells [11], which might be linked with some hematological malignancies.

The long latency and distinct multistage process of malignant cell transformation induced by the human mycoplasmas closely resemble human tumor development in nature. It has provided an excellent model system for studying molecular mechanisms of malignant cell transformation. Our earlier studies of possible molecular mechanisms revealed that over-expression of H-ras and c-myc oncogenes was associated with malignant transformation of C3H cells [12]. On the other hand, no evidence of integration of mycoplasmal genetic material (DNA) could be found in the malignant C3H cells induced by mycoplasmal infections [13]. Since many different genes are likely involved in the malignant cell transformation, a global analysis of the gene expression at the different stages of mycoplasma-induced malignant transformation is highly desired. The recently developed cDNA microarray technology would allow us to effectively analyze the expression of many genes in a short period of time [14, 15]. This technology has been used to analyze gene expression in host cells infected with various microorganisms [16, 17], in virus transformed mammalian cells [18] and in cancer tissues [19]. In this study, we used the cDNA microarray technique to investigate the global changes of the gene expression in C3H cells at the different stages of the mycoplasma-induced malignant transformation. The array analyzed changes in expression of 1,185 different genes. Many of them have been proven to play an important role in cell malignant transformation.

The present study attempts to further elucidate the molecular mechanisms that may play a critical role in the complex malignant cell transformation process, using a mycoplasma-oncogenesis model we previously developed. The in vitro model system shares many important characteristics found in the development of human cancer, including long latency and multistages of progression [3]. The newly available microarray technique allows us to compare the expression profiles of more than 1000 genes that are likely to be associated with oncogenesis in cells at different stages of malignant transformation induced by mycoplasmal infection. Two groups of molecules, the oncogenes and tumor suppressor genes, and apoptosis-associated genes, which could be of particular importance in the development and progression of malignant tumors, were included in this study.

At the early stage of mycoplasmal infection (the 1st week), C3H cells responded with a gene expression profile often seen in cells following acute microbial infections [22–25], many more genes with increased expression than genes with decreased expression. As a result, a significant left shift of the correlation line on the scatter graph of the gene expression was noted in Fig. 3. About half of the over-expressed genes were related to cell signaling (receptors, cell signaling and intracellular transducers/effectors/modulators, Table 1). Thus, despite the seemingly unapparent infections of mycoplasmas in culture (and in vivo), mammalian host cells evidently had a strong response and produced a host of signaling molecules to coordinate messages that were important for the cells' adaptation to the new environment.

C3H cells at the reversible stage of transformation, following 11 weeks of mycoplasmal infection, were apparently in a transitional phase between benignancy and malignancy. Eradicating the transforming mycoplasmas from the culture with antibiotics could reverse all the malignant phenotypes of C3H cells. The gene expression profile of C3H cells at this specific stage was more volatile and appeared to reflect a transitional phase with more genes having aberrant expression than at any other stages during the mycoplasma-induced malignant transformation. On the other hand, a comparison of gene expression levels between cells at the early mycoplasma infection stage and at the reversible stage revealed that the correlation line was at nearly 45° and expression signatures (dots) were distributed relatively close to the line (Fig. 3D). This scatter distribution of the gene expression signatures suggested that the gene expression pattern at the reversible stage in general still mostly resembled that at the early mycoplasma infection stage. Many studies have shown that malignant phenotypes induced by quantitative changes of oncogene(s) could be reversed by interrupting the activation of causative oncogene(s) [26]. Our earlier study revealed that eradication of mycoplasmas from cell cultures with an antibiotic would abolish the aberrant expression of c-myc and H-ras and reverse all of the malignant properties of C3H cells at this stage [12]. These data support our theory that malignant phenotypes of the cells at the reversible stage might be caused by quantitatively aberrant expression of certain oncogenes and tumor suppressors in the response to chronic mycoplasmal infection.

C3H cells became irreversibly or permanently transformed following 18 weeks of mycoplasmal infection and gained the ability to form tumors in animals and colonies in soft agar. These cells were no longer dependent on the continuing presence of mycoplasmas for maintaining their malignant properties [3]. Our present study revealed that cells at this stage had the highest gene expression rate among the 3 different stages in the transformation process (Table 1). However, the number of genes with altered expression actually decreased 27% from that at the reversible stage (Table 1). A comparison of gene expression profiles between control cells and cells at the 3 three progressive stages of the transformation showed that the correlation lines on the scattered gene expression graphs were gradually right-shifting (Fig. 3A, 3B and 3C). The correlation coefficients were gradually decreasing. Although the correlation line in the gene expression scatter plot between the control cells and cells at the irreversible stage was near 45° (Fig. 3C), the expression signatures were more scatter than those at any other stages of the transformation process. These collective results suggest that the gene expression profile at the irreversible stage might be changed from a volatile status at the reversible stage to a relatively stable status that is far more different from that of the noninfected control cells. The newly established gene expression pattern at the irreversible stage may provide essential material basis for a new set of interactions of molecules, which support the stable malignant phenotypes of the transformed cells.

The cells at the irreversibly transformed stage also had the highest expression rate of cancer-related genes during the mycoplasma-induced malignant transformation (Table 1). Increase of oncogene expression and decrease of tumor suppressor genes expression at the reversible stage (Table 2) seems to provide a relatively straightforward explanation for the mycoplasma mediated malignant transformation. However, we also found decreased expression of 4 oncogenes at the reversible stage and increased expression of one tumor suppressor at the irreversible stage. A similar phenomenon was also observed in the expression profile of apoptosis associated genes (Data not shown). Mutation(s) in these molecules could be responsible for the decreased oncogene expression and increased tumor suppressor gene expression in the transformed cells. The mutation could alter physiological functions of tumor- and apoptosis-associated genes. The host cells might compensate the normal functions of the affected molecules by adjusting their expression levels. This might be the case in the mycoplasma-induced transformation. Chronic mycoplasmal infection could cause the genetic instability and changes in the genetic material (DNA) of host cells [2, 3, 11].

There are tens of thousand of different molecules and hundreds of biochemical pathways in mammalian cells. Over-expression or down-regulation of a single molecule that plays an important role in the control of cell proliferation could evidently alter the balance of the entire cell physiological network and lead to malignant transformation [27, 28]. We believe that prolonged over-expression and/or down-regulation of a group of important molecules would likely change the critical balance of physiological networks in cells chronically infected by mycoplasmas and result in malignant cell transformation. The present microarray study has allowed us to identify changes of expression (up-regulated and down-regulated) of many important oncogenesis-associated genes at the different stages of mycoplasma-mediated cell transformation. We believe that the pattern of gene expression profile alterations observed in our mycoplasma-induced transformation model might be similar to those found in many transformation models induced by transforming microorganisms. However, due to the differences between the transforming microorganisms, alterations of many individual genes observed in this study could be unique to the mycoplasma-induced transformation process. Therefore, more work is needed to identify genes specific for mycoplasma-induced transformation and which genes are truly responsible for the progression of this complex process. The further challenge is to elucidate how the aberrant expression of these genes in cells would cause permanent or constitutive activation of certain oncogenes and leads to the irreversible malignant transformation.

This study reveals different gene expression profiles of C3H cells at different stages of the mycoplasma-induced malignant transformation. The results indicate that these expression profiles correspond with the phenotypic features of the cells in the mycoplasma induced transformation process. The early mycoplasma infection stage shares a common phenomenon in the gene expression profile with many other acute infections, genes with increased expression outnumbering those with decreased expression. The reversible stage is relatively unstable and chaotic with the highest number of altered genes. Its profile reflects the nature of the transition between benignancy and malignancy at the molecular level. The expression profile at the irreversible stage shows a unique pattern of a wide range of expression levels and an increased number of expressing genes, especially the cancer-related genes. Aberrant expression of oncogenes and tumor repressor might play a key role in the mycoplasma-induced malignant transformation.