Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells

Abstract

Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including those of the prostate. The American Cancer Society, estimates that approximately 20% of all worldwide cancers are caused by infection. Mycoplasma, a genus of bacteria that lack a cell wall, are among the few prokaryotes that can grow in close relationship with mammalian cells, often without any apparent pathology, for extended periods of time. In this study, the capacity of Mycoplasma genitalium, a prevalent sexually transmitted infection, and Mycoplasma hyorhinis, a mycoplasma found at unusually high frequency among patients with AIDS, to induce a malignant phenotype in benign human prostate cells (BPH-1) was evaluated using a series of in vitro and in vivo assays. After 19 weeks of culture, infected BPH-1 cells achieved anchorage-independent growth and increased migration and invasion. Malignant transformation of infected BPH-1 cells was confirmed by the formation of xenograft tumors in athymic mice. Associated with these changes was an increase in karyotypic entropy, evident by the accumulation of chromosomal aberrations and polysomy. This is the first report describing the capacity of M. genitalium or M. hyorhinis infection to lead to the malignant transformation of benign human epithelial cells and may serve as a model to further study the relationship between prostatitis and prostatic carcinogenesis.

Figure 1. Experimental plan.

Benign human prostate BPH-1 cells were infected with the mycoplsmas M. genitalium or M. hyorhinis and maintained in continuous tissue culture for 20 weeks. Uninfected BPH-1 cells were cultured in parallel for comparison. At weeks 4, 7, 11, 15 and 19, mycoplasmal infection status was confirmed by PCR, and a series of in vitro assays were performed. In vitro assays included anchorage-independent growth in soft agar, and migration and invasions assays using modified Boyden chambers. At weeks 7 and 19, infected and uninfected BPH-1 cells were inoculated into athymic mice to assess tumorigenicity

Figure 2. In vitro invasion and migration.

BPH-1 cells with and without M. genitalium (Mg) or M. hyorhinis (Mh) infection were tested for migration (A) and invasion (B) potential at weeks 7 and 19 post-infection. Both M. genitalium and M. hyorhinis infected BPH-1 cells had increased migration and invasion rates at week 19. Data presented are means of triplicates, and the error bars represent the standard error of the mean. *, p<0.05 versus no mycoplasma control (M-).

Figure 3. Anchorage-independent growth.

BPH-1 cells infected with M. genitalium (Mg) or M. hyorhinis (Mh) were tested for the ability to grow in soft agar. Persistent infection for 19 weeks with either mycoplasma induced anchorage-independent growth of BPH-1 cells. The PC-3 prostate cancer cell line was used as a positive control. (10 micrometer scale bar  = 2 mm)

Figure 4. Cytogenetic analysis of mycoplasma infected BPH-1 cells.

Metaphase chromosomal complement was revealed by spectral karyotyping (SKY) and counter-staining with DAPI. Representative metaphase cells after SKY classification are shown. A, uninfected BPH-1 cells. B, BPH-1 cells after 19 weeks M. hyorhinis infection. Composite for each chromosome shows, left to right, inverted DAPI-stained image, and spectral imaging color representation. Chromosome number is shown below image groupings, numbers alongside the derivative chromosomes indicate the origin of the translocated material. M. hyorhinis exposed BPH-1 cells were noted to harbor trisomy of chromosomes 1, 2, 6, 7, 15–18, and 21, in addition to loss of chromosome 13.

Figure 5. Molecular analysis of human BPH-1 xenograft tumors grown in nude male mice.

Tumors generated by mycoplasma infection in BPH-1 cells did not have a typical adenocarcinoma appearance, but a more squamoid appearance (H&E). In addition, uninfected BPH-1 cells from passage 19 grown subcutaneously in nude mice were less angiogenic (CD34) and harbored more leukocytes (CD45) than did xenograft tumors from BPH-1 cells infected with M. hyorhinis or M. genitalium. Only mycoplasma infected BPH-1 cells formed xenograft tumors. Expression of markers of malignancy (CD44v9 and Ki-67) were revealed using immunohistochemistry. (10 micrometer scale bar  = 2 mm)