The study material consisted of 83 blocks of paraffin-embedded LSCC samples (51 cases from Pathomorphology Division of J. Babiński Regional Hospital in Wroclaw and 32 cases from Department and Clinic of Otolaryngology, Head and Neck Surgery, Wroclaw Medical University), operated and treated in 1997–2003. Ten sample blocks of benign hypertrophic lesions of laryngeal epithelium were used as a control (vocal cord nodules and Reinke's oedema). Patients were observed for 156 months, and 46 of them died during this period (55%). During the treatment, the age of patients was in the range of 39–79 years (average age, 60 years). Of all patients, 12 were women and 71 were men. Grade of malignancy (G) and clinical stage of disease were determined based on TNM classification (41). Clinical and pathological characteristics of patients are shown in Table I.

Immunofluorescence (IF) experiments and western blot analyses were conducted with the use of reference adherent laryngeal carcinoma cells line, Larynx Epidermoid Carcinoma HEp-2 (collection of cell lines of Ludvik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland). The cells were cultured in MEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, streptomycin and L-glutamine. Pooled primary human foreskin keratinocytes (Gibco, Paisley, UK) cell line was used as a control. The cells were cultured on Keratinocyte-SFM medium (Gibco) with L-glutamine. SFM (Gibco) supplement containing human growth factors (EGF) and bovine pituitary extract (BPE) was added to the medium. After thawing, the cells were passaged three times before being used for research. Cultures were carried out in HERA cell (Heraeus, Hanau, Germany) incubator under constant conditions: temperature of 37°C, CO₂ concentration of 5% and a 95% level of humidity.

Specimens of laryngeal carcinomas and benign lesions were fixed in 10% buffered-formalin, dehydrated and paraffin-embedded. In each case, histopathological evaluation and determination of LSCC grade was performed by haematoxylin and eosin (H&E) staining by two pathologists. In order to prepare H&E staining, paraffin blocks were cut into 7 μm thick section with the use of RM 2145 microtome (Leica Biosystem, Nussloch, Germany). For IHC reaction, paraffin blocks were cut in sections 4 μm thick, which were placed onto Superfrost Plus slides (Menzel Gläser, Braunschweig, Germany). Deparaffinisation, hydration and heat induced epitope demasking were carried out by boiling for 20 min in the temperature of 97°C in Dako PT Link (Dako, Glostrup, Denmark) apparatus in high pH Target retrieval solution (Dako), according to 3-in-1 procedure. Endogenous peroxidase activity was blocked by incubation with peroxidase-blocking solution (Dako). For the evaluation of expression level of MCM2, MCM3, MCM7, Ki-67 antigen and MT-I/II in LSCC cells, IHC reactions were carried out in paraffin sections with the use of specific monoclonal mouse anti-human antibodies: anti-Ki-67 (dilution 1:100; clone MIB1, code no. F7268; Dako), anti-MT-I/II (dilution 1:100; clone E9, code no. M0639; Dako), anti-MCM2 (dilution 1:15; clone CRCT2.1, code no. NCL-MCM2; Leica Biosystems); anti-MCM3 (dilution 1:50; clone 101, code no. M7263; Dako); anti-MCM7 (dilution 1:50; clone DCS-141.1, code no. NCL-MCM7; Leica Biosystems). The antibodies were diluted in background-reducing reagent and the incubation with sections was carried out for 1 h in room temperature. All IHC reactions were carried out with the use of Autostainer Link 48 (Dako) apparatus and EnVision™ FLEX reagents (Dako) visualisation system. Sites, where a given antigen was located, were visualized in IHC reactions by brown DAB (3,3′-diaminobenzidine tetrahydrochloride) staining. Omitting the addition of primary antibody was used for negative control of IHC reaction, whereas LSCC cases characterised by high expression of studied markers served as a positive control.

Two pathologists carried out the evaluation independently. The estimation of nuclear expression of MCM2, MCM3, MCM7 and Ki-67, antigens was performed at ×200 magnification with the use of BX41 (Olympus, Tokyo, Japan) light microscope coupled with visual circuit and Cell^D (Olympus) software for computer image analysis. The intensity of MCM2, MCM3, MCM7 and Ki-67 expression was determined with the use of five-point evaluation scale (score 0–4), taking into account the percent of cancer cells exhibiting reaction relative to all cancer cells in a given specimen (Table II) (38,42). For the evaluation of MT-I/II cytoplasmic expression level, semiquantitative IRS (method according to Remmele and Stegner - immunoreactive score) was used (43). The intensity of colour reaction (score 0–3) and percentage amount of IRS-positive cancer cells (score 0–4) was estimated. The final result was the product of scores obtained for the evaluation of both parameters and values from 0 to 12 were considered (Table III).

After 72 h of culturing, the cells were trypsinised, centrifuged and PBS-washed. The number of cells was estimated using Burker's chamber. For each western blot analysis, 3.5–4×10⁶ of laryngeal cancer cells and keratinocytes in exponential growth phase were taken. After washing with cold PBS, the cells were resuspended in 1 ml of isotonic buffer for cell nuclei isolation (5 mM MgCl₂, 50 mM NaCl, 50 mM Tris-HCl pH 7.5, 250 mM sucrose). Cells were mechanically broken on ice in isotonic buffer, in glass Potter's homogeniser. In order to separate nuclei pellet from cytoplasm, homogenate was centrifuged for 10 min in 4°C, 2000 × g. Nuclei precipitate was solubilised on ice using nuclei lysis buffer [50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.1% Igepal-NP-40 (Sigma, St. Louis, MO, USA), protease and phosphatase inhibitors (Sigma), 0.5 mM PMSF]. The suspension was centrifuged for protein separation. Supernatant was stored in −20°C. For the determination of concentration of obtained protein, BCA method was used (Thermo Fisher Scientific-Pierce, Rockford, IL, USA). Cellular extracts were mixed with GLB sample buffer (250 mM Tris pH 6.8, 40% glycerol, 20% β-mercaptoethanol, 100 mM DTT, 0.33 mg/ml bromophenol blue, 8% SDS) and then denatured for 10 min in 95°C. Equal amounts of proteins from cancer cells and normal keratinocytes were loaded into a gel (15 μg/lane) and separated by electrophoresis in 7.5% polyacrylamide gel with SDS in Mini-Protean 3 apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Next, the proteins were electrophoretically transferred onto PVDF-Immobilon P (Millipore, Billerica, MA, USA) membrane according to Towbin et al (44) and the membrane was blocked with milk solution in TBS with 0.1% Tween-20 (TBST) for non-specific binding sites.

The expression of MCM2, MCM3 and MCM7 was determined using specific mouse monoclonal anti-human antibodies: anti-MCM2 (dilution 1:100; clone CRCT2.1, code no. sc-56321; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MCM3 (dilution 1:400; clone 36-Q-7, code no. sc-81848; Santa Cruz Biotechnology); anti-MCM7 (dilution 1:200; clone 141.2, code no. sc-9966; Santa Cruz Biotechnology). Incubation was carried out overnight at 4°C with mild shaking. The membrane was washed three times using 0.2% TBST buffer. Afterwards, it was incubated in the solution of donkey anti-mouse antibody conjugated with horseradish peroxidase (1:3000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The detection was performed with the use of chemiluminescence substrate from Immun-Star HRP Chemiluminescent kit (Bio-Rad Laboratories). The data were collected at various exposition times ranging from 2 sec to 30 min in Chemi-Doc XRS Molecular Imager (Bio-Rad Laboratories) apparatus. Antibodies were washed from the membrane to perform densitometric studies and the detection of β-actin was conducted with the use of mouse monoclonal primary antibody (dilution 1:300; clone AC-40, code no. A4700; Sigma, St. Louis, MO, USA) in SNAP i.d (Millipore) apparatus, as a control of amount of loaded protein. Quantity One (Bio-Rad Laboratories) software was used for measurements of the protein band optical density. In each lane, the amount of protein in MCM2, MCM3 and MCM7 bands was normalised to β-actin.

In order to perform immunofluorescence reactions on cells of laryngeal cancer cell line HEp-2 and normal human keratinocytes, 24-h micro-cultures were set up on slides with 8-wells covered with Teflon. The cells were trypsinised, centrifuged and resuspended in 5 ml of medium. For microculture inoculum, 50 μl of 5×10⁴ cells/ml suspension was instilled into wells on the slide. Slide-microcultures were placed on Petri dishes in an incubator at 37°C for 24 h. The medium was removed after the incubation and wells were washed with PBS. The cells were fixed in wells with the use of 4% formaldehyde in PBS for 12 min at room temperature. Cell membrane permeabilisation was performed with 0.2% Triton in PBS for 10 min at room temperature. Specific primary mouse monoclonal antibodies were used for the reaction: anti-MCM2 (dilution 1:15; clone CRCT2.1, NCL-MCM2; Leica Biosystems), anti-MCM3 (dilution 1:20; clone 101, M7263; Dako), anti-MCM7 (dilution 1:50; clone DCS-141.1, NCL-MCM7; Leica Biosystems). Overnight incubation with primary antibodies was carried out at 4°C. Next, preparations were incubated for 1 h with donkey anti-mouse secondary FITC-conjugated antibody (Jackson ImmunoResearch Laboratories) diluted 1:50 in the reagent with background-reducing component. Preparations were mounted using Vectashield Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA), which contains DAPI for cellular DNA visualisation. Observations were made at ×200 magnification with the use of BX51 microscope (Olympus) coupled with Cell^F software (Olympus).

Statistical analyses were performed using Prism 5.0 (Graphpad Software Inc., La Jolla, CA, USA) and Statistica 8.0 (Dell Software, Aliso Viejo, CA, USA) software. In order to study the relationship between the antigens, Spearman's rank correlation was used. Mann-Whitney U test, a non-parametric equivalent of Student's t-test, as well as Kruskal-Wallis test, a non-parametric equivalent of variation analysis, were used for the evaluation of relationship between the intensity of gene expression and LSCC grade. Fisher's exact test was used to determine the correlation between the intensity of marker expression and clinical and pathological factors. To verify the correlation between protein expression levels and the patient survival, Kaplan-Meier method and Cox proportional hazards regression analysis were used. Statistically significance was set at P-value <0.05.

Nuclear expression of MCM2, MCM3, MCM7 and Ki-67, as well as cytoplasmic-nuclear MT-I/II expression in LSCC was observed (Fig. 1) in histopathological specimens prepared by IHC. Strong MCM2 protein expression was reported in 47 (56.6%) cases of LSCC. Similarly high MCM3 expression was observed in 50 (60.2%) cases, while high MCM7 expression was recorded in 48 (57.8%) cases. Intensity of Ki-67 expression in laryngeal cancer cells was weaker. High expression was reported in 39 (46.9%) cases of LSCC. Higher expression of all tested MCM proteins, as well as Ki-67 and MT-I/II in LSCC was visible in comparison to analysed benign lesions (Fig. 1). Statistical analysis revealed that this difference is statistically significant in the case of each of proteins being evaluated (Table IV).

Spearman's rank correlation coefficient showed strong positive correlation between the level of MCM2, MCM3 and MCM7 expression and Ki-67 antigen (r=0.60, r=0.52, r=0.54; P<0.05) in LSCC (Table V). The strongest correlation was observed between MCM2 and Ki-67. Additionally, moderate positive correlation between expression of MCM3 and MT-I/II was found (r=0.35, P<0.05). No statistically significant correlation was found between expression of MT-I/II and two other MCM proteins (Table V).

The greater the level of expression of MCM2, MCM3, MCM7 and Ki-67 marker was in cancer cells, the higher was the grade (G) of the analysed LSCC. Additionally, statistically significant difference was shown between G1 and G3 in the level of expression of MCM3, MCM7 and Ki-67 proteins, and between G1 and G2 in the case of MCM2 and MCM7, and between G2 and G3 in the case of MCM2 and Ki-67 (Table VI and Fig. 1). The difference in MCMs, Ki-67 and MT-I/II expression was observed in cases of LSCC with and without lymph node metastasis. The difference was statistically significant in the case of MCM2 and Ki-67 (Table VII).

No significant difference was found in the survival of patients with high (score 3–4 for MCM 2, 3, 7 and Ki-67; score 6–12 for MT-I/II) and low (score 0–2 for MCM 2, 3, 7 and Ki-67; score 0–4 for MT-I/II) expression of studied proteins, neither was the survival dependent on the age of the patients. However, significant relationship was shown between patient survival and lymph node metastasis, as well as the size of the tumour (Table VIII).

In order to visualise the localisation and intensity of expression of MCM2, MCM3 and MCM7 in HEp-2 cells and normal keratinocytes, in vitro studies were performed. IF reactions were conducted with the use of specific antibodies on slide-microcultures. Next, fluorescence of nuclei in HEp-2 cells and normal keratinocyte cell lines was observed under the microscope (Fig. 2). Importantly, larger number of cells was expressed in the studied MCM proteins in laryngeal cancer cells than in normal keratinocytes.

MCM2, MCM3 and MCM7 proteins were detected in nuclei isolates of HEp-2 cell line and keratinocytes by means of western blot technique and with the use of specific antibodies. Densitometrically measured level of expression of MCM2, MCM3 and MCM7 was higher in HEp-2 cells in comparison with their expression in keratinocytes (Fig. 3).