In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo

Abstract

Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxia-induced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia (4 h) three times a week. The resulting two hypoxia-conditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxia-conditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient (SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs.

Figure 1

Survival of conditioned cells following various periods of hypoxia, as measured by the MTT assay. Cells were exposed to hypoxia for 4 h three times weekly, for up to 40 exposures (corresponding to HP10 to HP40 and HCP10 to HCP40), and cells harvested and stored after every 10 hypoxic treatments. For analysis of survival in hypoxia, cells were plated in 96-well plates, exposed to profound hypoxia for the durations indicated, and cultured under oxic conditions for an additional 72 h.

Figure 2

Sensitivity of hypoxia-conditioned cell lines to cisplatin as measured by the MTT assay. Cells were plated in 96-well plates, and after 18–24 h to allow adherence, a range of concentrations of cisplatin was added. For hypoxic incubation, the plates were immediately placed in the anaerobic chamber, left for 24 h at 37°C, and returned to an oxic environment for an additional 48 h. The MTT assay was performed at 72 h after initial exposure to the drugs in all cells. The one asterisk denotes the statistically significant difference between hypoxia-conditioned cell lines and its parental cell line with P-value <0.05.

Figure 3

Tumour growth curve of HT29, HP40 (A), and HCT116, HCP40 (B) in SCID mice. tumours were generated by subcutaneous injection of 10⁷ cells in the left flank, and bidimensional measurements made every three days. The curves shown are the combined results of two separate experiments, the results of which were comparable.

Figure 4

(A) Northern blot of RNA from the cell lines indicated, under oxic or hypoxic (16 h) conditions, probed with human VEGF and HIF-1α cDNAs. (B) Immunohistochemistry of formalin-fixed, paraffin-embedded tumour sections following 7 days' tumour growth of HT29, HP40, HCT116 and HCP40 implanted subcutaneously in scid mice. Sections of tumour were incubated with rat anti-mouse CD31, and developed by an immunoperoxidase technique.