Tumor environmental factors glucose deprivation and lactic acidosis induce mitotic chromosomal instability--an implication in aneuploid human tumors

Abstract

Mitotic chromosomal instability (CIN) plays important roles in tumor progression, but what causes CIN is incompletely understood. In general, tumor CIN arises from abnormal mitosis, which is caused by either intrinsic or extrinsic factors. While intrinsic factors such as mitotic checkpoint genes have been intensively studied, the impact of tumor microenvironmental factors on tumor CIN is largely unknown. We investigate if glucose deprivation and lactic acidosis--two tumor microenvironmental factors--could induce cancer cell CIN. We show that glucose deprivation with lactic acidosis significantly increases CIN in 4T1, MCF-7 and HCT116 scored by micronuclei, or aneuploidy, or abnormal mitosis, potentially via damaging DNA, up-regulating mitotic checkpoint genes, and/or amplifying centrosome. Of note, the feature of CIN induced by glucose deprivation with lactic acidosis is similar to that of aneuploid human tumors. We conclude that tumor environmental factors glucose deprivation and lactic acidosis can induce tumor CIN and propose that they are potentially responsible for human tumor aneuploidy.

Figure 1. Lactic acidosis rescues HCT116, 4T1 and MCF-7 cells from glucose deprivation.

4T1 or MCF-7 cells were cultured in RPMI-1640 containing 3 mM glucose with or without lactic acidosis. HCT116 cells were cultured in RPMI-1640 containing 0.5 mM glucose with or without lactic acidosis. Cell count, lactate and glucose in culture medium were determined as described in Materials and Methods. (A) (B) & (C) Curves of cell growth/death, glucose consumption, and lactate generation. Solid symbol, with lactic acidosis; open symbol, without lactic acidosis. * p<0.05, **, p<0.01, *** p<0.005, as compared with control.

Figure 2. One cycle of glucose deprivation with lactic acidosis followed by nutrient restoration significantly increases micronuclei (MNi), nucleoplasmic bridges (NPBs), and nuclear blebs (NBs) in HCT116, 4T1 and MCF-7 cancer cells.

(A) Percentage of binucleated (BN) cells in the cells that were cultured for 7 days as described in Figure 1 and resume mitosis upon nutrient restoration in the presence of cytochalasin B. Control cells are maintained in regular medium without lactic acidosis. (B) The numbers of cells that carry MNi, or NPBs, or NBs in every 1000 BN cells. *p<0.05, **p<0.01, *** p<0.005, in comparison to control. MNi, NPBs and NBs were determined by cytokinesis block micronucleus (CBMN) assay as described in Materials and Methods. (C) Representative photos of MNi, NPBs, and NBs. (D) MNi that contains chromosomes by pancentromeric FISH probes. MNC+, MNi containing whole chromosome; MNC−, MNi containing chromosomal accentric fragment.

Figure 3. The effect of lactic acidosis, glucose deprivation, and lactic acidosis with glucose deprivation on chromosome instability.

(A) The numbers of HCT116 cells that carry MNi, or NPBs, or NBs in every 1000 BN cells. *p<0.05, **p<0.01, *** p<0.005. HCT116 cells were cultured in 4 different conditions: condition 1 (control), regular RPMI-1640; condition 2 (glucose deprivation), RPMI-1640 containing 0.5 mM glucose; condition 3 (lactic acidosis), regular RPMI-1640 with 20 mM lactic acidosis. To avoid glucose deprivation during 7-day culture, we replaced the medium every 2 days; condition 4 (glucose deprivation with lactic acidosis), RPMI-1640 containing 0.5 mM glucose with 20 mM lactic acidosis. Cells were harvested at the indicated time and subjected for analysis of MNi, or NPBs, or NBs. (B) The numbers of 4T1 cells that carry MNi, or NPBs, or NBs in every 1000 BN cells. *p<0.05, **p<0.01, *** p<0.005, in comparison to control. 4T1 cells were cultured in 4 different condition as described in (A) except condition 2 and 4 in which glucose was 3 mM.

Figure 4. One cycle of glucose deprivation with lactic acidosis followed by nutrient restoration significantly increases aneuploidy in HCT116 cells.

Cells were cultured in RPMI-1640 medium containing 0.5 mM glucose with lactic acidosis for 7 days. The cells surviving through glucose deprivation were then cultured in fresh medium for 48 hours and subjected for aneuploid analysis. Control cells were maintained in regular RPMI-1640 medium without lactic acidosis. (A) Representative photos of diploid and aneuploid cells scored by chromosomes 7(green) and 17(red). (B) Percentage of aneuploid cells scored by chromosome 7 or 17. *p<0.05, **p<0.01, as compared with control. The results were confirmed by 2 independent experiments.

Figure 5. One cycle of glucose deprivation with lactic acidosis followed by nutrient restoration induces centrosome amplification and multipolar division in 4T1 cells.

Cells were cultured in RPMI-1640 medium containing 3 mM glucose with lactic acidosis for 7 days. The cells surviving through glucose deprivation with lactic acidosis were then cultured in fresh medium for 48 hours and subjected for analysis of centrosome and spindle. Control cells were maintained in regular RPMI-1640 medium without lactic acidosis. (A) Representative photos of nonmitotic cells with one or multiple centrosomes. (B) The percentage of cells with multiple centrosomes (>1) in the population of nonmitotic cells (pericentrin, green; DAPI, blue). **p<0.01, in comparison to control. (C) Representative photos of mitotic cells with bipolar or multipolar spindles. (D) The percentage of cells with multipolar spindles in the population of mitotic cells. **p<0.01, in comparison to control.

Figure 6. One cycle of glucose deprivation with lactic acidosis followed by nutrient restoration significantly increases multinucleation in 4T1 cells.

**p<0.01, in comparison to control. Cells were cultured in RPMI-1640 medium containing 3 mM glucose with lactic acidosis for 7 days. The cells surviving through glucose deprivation with lactic acidosis were then cultured in fresh medium for 48 hours. Control cells were maintained in regular RPMI-1640 medium without lactic acidosis.

Figure 7. The effect of glucose deprivation with lactic acidosis followed by nutrient restoration on key members of mitotic checkpoint in HCT116 cells.

Cells were cultured in RPMI-1640 medium containing 0.5 mM glucose with lactic acidosis for 7 days. The cells surviving through glucose deprivation with lactic acidosis were then cultured in fresh medium. Cells were collected at indicated time for real-time PCR (A) and Western Blot (B) analysis. The results were confirmed by 2 independent experiments.

Figure 8. The effect of glucose deprivation with lactic acidosis followed by nutrient restoration on cell cycle in HCT116 cells.

Cells were cultured in RPMI-1640 medium containing 0.5 mM glucose with lactic acidosis for 7 days. The cells surviving through glucose deprivation with lactic acidosis were then cultured in fresh medium. (A) & (B) Cells were collected at indicated time for analysis of cell cycle and phospho-Histone H3 labelling analysis. (C) The cell growth after release from stress upon nutrition restoration. The results were confirmed by 2 independent experiments.

Figure 9. The effect of glucose deprivation with lactic acidosis followed by nutrient restoration on DNA damage in HCT116 cells.

Cells were divided into 3 groups. Group 1 (control), cells were cultured in regular RPMI-1640 medium; Group 2 (glucose deprivation with lactic acidosis), cells were cultured in RPMI-1640 medium containing 0.5 mM glucose with lactic acidosis for 7 days; Group 3 (nutrition restoration), cells were cultured in RPMI-1640 medium containing 0.5 mM glucose with lactic acidosis for 7 days then cultured in fresh medium for 48 hours. DNA damage was scored by γ-H2AX and 53BP1 labelling as described in Materials and Methods. (A) Representative photos of cells with γ-H2AX and 53BP1 labelling. (B) Number of γ-H2AX per cell (n = 300) and statistical analysis. (C) Number of 53BP1 foci per cell (n = 300) and statistical analysis. *p<0.05, **p<0.01, in comparison to control.