Expression of NMHC IIs at mRNA in the 3MC-induced tumor in mice

Recent studies reported that 3MC either induces the expression of cytochrome P4501A1, P4501A2, UDP-glucuronosyl transferases in rats and expression of multiple drug resistance genes 1 in a hepatoma cell line Hepa-1c1c7 [5,24] or reduces the expression of genes such as muscle specific creatine kinase in mice [2]. Given the role of NM IIs in cell division, we decided to investigate the expression of NM II isoforms in 3MC-induced tumor tissue (T) by reverse transcription and polymerase chain reaction amplification (RT-PCR), using primers specific for each heavy chain. Figure 1A shows that mRNA of NMHC II-A and II-B is expressed at a higher level in tumor cells compared with normal tissue associated with the tumor (NTAT), as shown by the generation of a nucleotide fragment of 254 bp for II-A, 289 bp for II-B. On the other hand, NMHC II-C has two mRNAs in the tumor tissue as two fragments of 190 bp and 214 bp were seen using primers flanking the loop 1 splicing site in the head region of its heavy chain, similar to a previous finding by Golomb et al. [13]. Quantification by real-time PCR (Fig. 1B) shows a 25.7- and 19.03-fold induction in the expression of NMHC II-A and II-B mRNA, respectively, in tumor tissue compared with NTAT. In contrast, although tumor tissue expressed both II-C0 (190 bp) and II-C1 (214 bp), quantification of NMHC II-C mRNA by real-time PCR (Fig. 1B) revealed no statistically significant difference with normal tissue, which expressed only II-C0. Other alternatively spliced isoforms like NMHC II-B1, NMHC II-C2 and NMHC II-B2, whose expressions are neuronal specific [16,17], were not detected in 3MC-induced tumor tissue (data not shown).

Expression of NMHC II protein in the 3MC-induced tumor in mice

We were therefore interested to see the protein level of NM IIs in 3MC-induced tumor tissue. We quantified the relative difference in protein expression of NMHC IIs between tumor and NTAT using antibodies specific for each isoform. Figure 2A shows immunoblots probed with antibodies to NMHC II-A and II-B. Figure 2B quantifies the relative expression of each isoform, considering the quantity of NMHC II in the NTAT as ‘1’ for each isoform. We performed a series of different immunoblots (n ≥ 3 from each mouse group); one representative blot is shown in Fig. 2A. The protein expression analysis correlates with the increase in mRNA expression (Fig. 1A) and shows a 32.96- and 9.67-fold increase for II-A and II-B, respectively, in tumor tissue over the NTAT. In contrast, expression of NMHC II-C was undetectable at the protein level by immunoblot (Fig. S1).

We also analyzed the expression level of NMHC IIs with the progression of tumor growth. Unlike NMHC II-B, which is only detectable at a later stage of tumor formation (i.e. 96 days), NMHC II-A is detected on the seventh day after a second dose of 3MC (Fig. 3A). Figure 3B shows the quantification of NMHC II-A induction with time and dose of 3MC. Interestingly, no expression of NMHC II-A or II-B was seen in vehicle (olive oil) injected mouse tissue (Fig. 3C). Early expression of NM II-A may facilitate transformation of the normal cells to atypical and cancerous cells.

Mass spectroscopy analysis of NMHC IIs in normal and tumor tissue

To see the abundance of each NM II isoform in tumor and NTAT, we used mass spectroscopy analysis to quantify the peptide numbers for each of the NM II isoforms. Table 1 shows that 64.5 ± 3.5% II-A, 34 ± 3% II-B and 1.5 ± 0.25% II-C of total NM IIs are detectable in tumor tissue whereas no measurable amount of each of the NM II isoforms was detected in NTAT (n = 3 from each mouse group). Interestingly, although we were able to see NM II-C mRNA expression in tumor and normal tissues by RT-PCR analysis, the relative abundance of II-C at protein level was very low – only 1.5% in tumor or non-detectable in NTAT. These results suggest that NM II-A and II-B are more likely to be involved in transformation from normal cells to tumor cells and in rapid growth of transformed cells at the site of 3MC injection, compared with NM II-C.

Localization of NM IIs in 3MC-induced tumors in mice

In the 3MC mouse model, cellular transformation and tumor development occur exclusively at the site of carcinogen injection [8]. Our interest was to determine the localization of NM II-A and II-B in fibroblast cells which are thought to be transformed to atypical cells in tumors. We used isoform specific antibodies against the NMHC to examine the localization pattern in cells by immunofluorescence confocal microscopy. Hematoxylin and eosin staining of normal and tumor tissue confirmed the development of tumor at the site of 3MC injection (Fig. S2). Cells in the tumor tissue showed enlarged vesicular nuclei, prominent nucleoli and heterogeneous morphologies. Atypical fibroblast/tumor tissues expressed vimentin (a marker for fibroblast cells, red), NMHC II-A (green) and NMHC II-B (green) (bottom panels of Fig. 4A,B). In contrast, in normal tissue there were mainly two types of cell population: one population showed vimentin, NMHC II-A and NMHC II-B staining, which seemed to be fibroblast cells, and another population showed sarcomeric structure with NMHC II-A and II-B staining, albeit with less intensity compared with fibroblast cells (top panels of Fig. 4A,B). The quantification shown in Fig. 4C suggests more expression by 1.8-fold and 1.7-fold of NM II-A and NM II-B, respectively, in fibroblast cells compared with non-fibroblast cells in NTAT. Interestingly, expression of NM II-A and II-B remained the same in fibroblast cells from normal and tumor tissues. These data suggest that increased expression of NM II-A and II-B may be due to a greater number of fibroblast type cells in tumor tissue.

3MC induces the dedifferentiation of C2C12 myotubes in vitro

Although DNA structural changes and fibroblast transformation at the site of carcinogen injection are postulated to be the main causes for tumor formation in the 3MC-induced mouse tumor model, we wanted to test the possibility of dedifferentiation of muscle cells into mononucleated cells which can further develop tumors. We used an in vitro model system in which C2C12 myotubes were treated with 3MC. Figure 5A represents immunoblots in which expression of NM II-A and II-B in C2C12 myotubes treated with 20–50 nm 3MC (lanes 2, 3) or vehicle (lane 1) for 48 h was analyzed using antibodies specific for each isoform. Quantification of immunoblots (Fig. 5B) reveals that a 1.5 and 1.2 fold increase for II-A and II-B, respectively, in 50 nm 3MC treated myotubes over the vehicle or 20 nm 3MC treated myotubes. Note that II-C is undetectable in myotubes even in the presence of 3MC, which mimics the finding of no expression of NM II-C in the 3MC-treated mouse tumor model (data not shown).

When the myotubes were treated with 50 nm 3MC, images were taken every 24 h up to 144 h. At 48 h, 55% of the myotubes in the presence of 3MC showed cleavage furrows compared with 10% myotubes treated with vehicle (Fig. 5D). The average width of the myotubes was 30–45 μm, and places where the width was < 20 μm were considered as an example of cleavage furrow formation (arrows in Fig. 5C, part b, inset). We also used different concentrations of 3MC from 20 to 100 nm and treated with the carcinogen for different time periods from 24 to 144 h. Myotubes started to lose adhesion to the substratum or clumps of cells appeared at 72 h with a concentration of 3MC higher than 50 nm (data not shown). Figure 5C shows images of myotubes treated with vehicle (a) or 50 nm 3MC (b). This result suggests that myotubes can re-enter the cell cycle in the presence of the carcinogen, 3MC.

Development of tumor in the hind leg of mice

All animal experiments were carried out according to the guidelines of the Institutional Animal Ethics Committee. Appropriate measures were taken to minimize pain or discomfort for animals. 3MC-induced mouse tumor was generated using a previously reported method [2]. In brief, Swiss albino female mice were injected with a total of three doses of 3MC. Each dose was 10 mg·kg⁻¹ bodyweight in 0.1 mL of olive oil and was given at 1-week intervals. Mice were monitored for the appearance of growing tumor for the next 96 or 105 days. Tumor formation was confirmed by histological examination, in which muscle cells were replaced by cancerous cells. Solvent (olive oil) injected mice were used for controls, in which no tumor formation was observed.

RNA isolation, reverse transcription and quantitative real-time PCR

Total RNA from 3MC-induced tumor tissue and its associated normal tissue was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany). The total RNA (1 μg) was taken for cDNA preparation using random hexamers and the Gene-Amp RNA PCR core kit (Applied Biosystems, Branchburg, NJ, USA), and the resulting cDNA was amplified by PCR using primers specific to each of the NMHC II isoforms or by real-time PCR to quantify the amount of product using Syber green PCR master mix kit (Applied Biosystems). For C1 insert detection, primers were designed to the C1 flanking regions. The PCR program included four cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min and extension at 72 °C for 2 min, and then 31 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 2 min. The PCR products were analyzed by 1.8% agarose gel. For real-time PCR, the program includes an initial 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C for denaturation and 1 min at 60 °C for annealing and extension. After each cycle a melting curve analysis was performed to check that no primer dimer or non-specific products were formed. The primer sets were as follows: 5′-GCACATGTGGCCTCCTCACAC-3′ and 5′-ATGTGGAAGGTCCGCTCCTCT-3′ for mouse NMHC II-A; 5′-GCCCACGTTGCTTCTTCTCAC-3′ and 5′-CTGCTCCAGAGAGCAACTGAT-3′ for mouse NMHC II-B; 5′-GCCCATGTGGCATCATCTCCA-3′ and 5′-CTCCCACGATGTAGCCAGCA-3′ for mouse NMHC II-C, flanking to C1 exon; and 5′-GACAACTTTGGCATTGTGGAA-3′ and 5′-ACACATTGGGGGTAGGAACA-3′ for mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH was used to normalize the samples.

Electrophoresis and immunoblot analysis

As reported earlier [13], tissue extracts from mouse tissues were prepared using radioimmunoprecipitation assay (RIPA) buffer (50 mm Tris/HCl, pH 8.0, 150 mm sodium chloride, 1.0% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecylsulfate) with 5 mm ATP, 4 mm EDTA pH 8.0, 1 mm dithiothreitol, 10 mm MgCl₂, 0.5 mm phenylmethylsulfonyl fluoride and protease inhibitor cocktails (Sigma-Aldrich, St Louis, MO, USA) at 4 °C. The lysates were sedimented at 10 000 g for 10 min. The supernatant was fractionated by SDS/PAGE on 8% polyacrylamide Tris/glycine gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% skim milk with 0.05% Tween-20 in phosphate buffered saline. The upper part of the membrane was incubated overnight at 4 °C with antibodies to NMHC II-A (1 : 10 000, Cell Signaling Technology, Danvers, USA) and NMHC II-B (1 : 5000; Cell Signaling Technology). The lower part of the membrane was incubated with antibody to β-tubulin or β-actin (1 : 5000; Sigma-Aldrich). The membrane was washed and incubated with horseradish-peroxidase-conjugated secondary antibody (1 : 5000; Thermo Fisher Scientific Inc., Rockford, IL, USA, or Sigma-Aldrich) for 2 h at room temperature and developed with SuperSignal^® West Femto reagent (Thermo Fisher Scientific Inc.) or Immobilon Western reagent (Millipore Corporation, Billerica, MA, USA). Relative band intensity was quantified using imagej (National Institutes of Health, Bethesda, MD, USA) software after normalizing tubulin/actin band intensity.

Mass spectroscopy

Relative amounts of NM II isoform in tumor and normal tissue were estimated by mass spectroscopy, as previously reported [10,30,31]. Briefly, the tissue extract was fractionated by SDS/PAGE 6% polyacrylamide gels, and the gel was stained with Coomassie Blue. Bands near 230 kDa were excised followed by destaining, reduction and alkylation, and finally digestion with trypsin. Tryptic peptides were purified and concentrated on C18 resin Zip Tips (Millipore), and subjected to liquid chromatography tandem mass spectroscopy. Percentage contribution of each isoform to the total amount of NMHC II was calculated using the formula (n/N) × 100 where n stands for total peptide numbers generated from an isoform and N is the total peptide numbers generated from all NMHC II isoforms.

Immunostaining and imaging

The distribution of NMHC II isoforms in NTAT/T and its colocalization with vimentin were visualized by immunofluorescence staining using a previously reported method [32,33]. In brief, both T and NTAT were collected in phosphate buffered saline and then directly immersed in 4% paraformaldehyde overnight at room temperature. The fixed tissues were embedded in paraffin and sectioned at a thickness of 5 μm. For antibody staining, the samples were blocked with phosphate buffered saline containing 0.3% bovine serum albumin and 10% normal goat serum for 1 h at room temperature, incubated with polyclonal antibodies against NMHC II-A (1 : 1000; Cell Signaling Technology), II-B (1 : 1000; Cell Signaling Technology) and vimentin (1 : 100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 °C, followed by washes and then incubation with fluorescein-isothiocyanate-conjugated goat anti-rabbit IgG (1 : 200; Jackson Immuno Research, West Grove, PA, USA) and/or rhodamine-conjugated goat anti-mouse IgG (1 : 200; Santa Cruz Biotechnology Inc.) for 1 h at room temperature. After washing, the cover slips were mounted using a Prolong gold antifade reagent from Invitrogen. The images were collected using a C1 confocal microscope (Nikon, Tokyo, Japan).

Cell culture and myotube formation

Mouse myoblast cell line C2C12 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, according to ATCC standard protocols. Myotubes were produced by inducing fusion in myoblast cells at 70–80% confluence in differentiation medium containing 2% horse serum in DMEM for 5 days. Five days post differentiation, cells were treated with 20–100 nm 3MC or vehicle alone, dimethylsulfoxide, in a fresh differentiation medium containing 2% horse serum for another 1–6 days. After 48 h of 3MC treatment, cells were briefly washed twice with cold phosphate buffered saline and directly lysed with Laemmli sample buffer. Images were taken using an Olympus IX-51 microscope before the preparation of cell lysate.

Statistical analysis

Data were expressed as means ± SEM. Statistical significance was tested with a one-way analysis of variance followed by the Bonferroni test. P < 0.05 was considered to be significant.