The radiation response of cells recovering after chronic hypoxia

Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment.     

Inhibition of sphingosine kinase 1 suppresses proliferation of glioma cells under hypoxia by attenuating activity of extracellular signal-regulated kinase

Objectives: Sphingosine kinase (SphK), which is regulated by hypoxia, catalyses phosphorylation of sphingosine to produce sphingosine‐1‐phosphate, which stimulates invasiveness of gliomas. However, whether SphK is involved in proliferation of glioma cells under hypoxic conditions is not clearly understood. In this study, we have investigated the role of SphK in of proliferation glioma cells under hypoxia. Materials and methods: Effects of small interfering RNA (siRNA) on SphKs, SKI (inhibitor of SphK) and U0126 (inhibitor of ERK) on proliferation of glioma cells under hypoxia were studied using CCK‐8 assay and flow cytometry. Protein expression profiles were evaluated by Western blot analysis. Results:  SKI suppressed proliferation of glioma cells under hypoxia. Similarly, downregulation of SphKs by siRNA inhibited glioma cell proliferation, and the cell cycle was arrested in G₂/M phase when SphK1 was inhibited. In addition, inhibition of SphK1 attenuated phosphorylation of ERK in hypoxic conditions. Furthermore, U0126 markedly inhibited cell population growth and arrested cells in G₂/M as effectively as SKI. However, silencing SphK2 induced cell cycle arrest in the S phase and it showed little effect on hypoxia‐induced activation of ERK. Conclusions: SphK1 and SphK2 are involved in proliferation of glioma cells in hypoxic conditions through distinct signalling pathways. SphK1, but not SphK2, promotes cell population expansion in hypoxic conditions by activating ERK.     

Buoyant density of EMT6 fibrosarcoma cells: time course of the density changes after growth in hypoxia

EMT6 fibrosarcoma cells were grown to the exponential phase in tissue culture and incubated at 37 degrees C under hypoxic conditions. Buoyant density was determined as a function of the time in hypoxia. Hypoxia was produced in two ways. The first involved incubation of the cells in sealed aluminum chambers containing 95% N2, 5% CO2 gas, and < 10 ppm oxygen, resulting in the cells rapidly becoming exposed to the hypoxic environment. After incubation at 37 degrees C, they were centrifuged in linear Ficoll gradients to their isopycnic density. A significant decrease in density was found after 4 h, and prolonged incubation up to 24 h did not result in further change. This density change was reversible on transfer back to aerobic conditions, with the hypoxic cells reverting to their aerobic density after about 10 h reincubation in air. The second method of producing hypoxia involved growing about 8 X 10(6) cells in a medium-filled air-tight container. Hypoxia was produced gradually as the oxygen in the medium was consumed by cellular respiration. Similar results were obtained; that is, hypoxic cells became significantly less dense. However, when the level of hypoxia was varied between 4000 and < 10 ppm at 2-h intervals after the cells had depleted all of the original oxygen, no significant difference in density was found between hypoxic and aerobic cells.     

NA22598, a Novel Antitumor Compound, Reduces Cyclin D1 Levels, Arrests Cell Cycle at G1 Phase, and Inhibits Anchorage-Independent Growth of Human Tumor Cells

NA22598, a novel antitumor compound isolated from a microbial cultured broth, inhibited the growth of human colon cancer DLD-1 cells in suspension cultures (anchorage-independent growth) severalfold more strongly than in substratum-attached monolayer cultures. It arrested the cell cycle progression at early G1 phase under both these culture conditions. Rb phosphorylation, cyclin D1 expression, and cdk2 activation in G1 progression were all inhibited by NA22598, but the amounts of cdk2 and p27 were not affected. Among these effects the inhibition of cyclin D1 expression was most prominent, and NA22598 was found to inhibit the synthesis of cyclin D1 without affecting mRNA expression or protein degradation. p27 binding to cdk2 was more markedly increased in suspension cultures than in attached cultures by NA22598, but the compound had no effect on total p27. Apparently, the decrease of cyclin D1 induced redistribution of p27 from the cyclin D1/cdk4 to the cyclin E/cdk2 complexes during G1 phase in the suspension cultures. Because p27 is upregulated during suspension culture, a greater amount of it was associated with cyclin E/cdk2, thus producing greater growth inhibition. An agent, like NA22598, which induces the downregulation of cyclin D1 might offer a new anticancer strategy.     

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5 mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (?1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5 mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (?1% oxygen) and hypoxia mimetic CoCl₂. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5 mM glucose after 48 h of physical hypoxia. However, combining the latter with 100 μM CoCl₂ was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl₂ in 5 mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.     

Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.     

p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.     

Hypoxia-Induced Apoptosis in Human Cells with Normal p53 Status and Function, without Any Alteration in the Nuclear Protein Level

We have studied hypoxia-induced inactivation of cells from three established human cell lines with different p53 status. Hypoxia was found to induce apoptosis in cells expressing wild-type p53 (MCF-7 cells), but not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosis was demonstrated by DNA fragmentation, using agarose gel electrophoresis of DNA and DNA nick end labeling (TUNEL). We demonstrate that extremely hypoxic conditions (<4 ppm O₂) do not cause any change of expression in the p53 protein level in these three cell lines. In addition, the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic conditions. Furthermore, no correlation was found between the p53-expression level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis occurred in all phases of the cell cycle, although predominantly for cells in S phase. Apoptosis was observed only during a limited time window (i.e., ≈10 to ≈24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to form visible colonies following 15 h exposure to extreme hypoxia, only ∼28% were induced to apoptosis, suggesting that ∼38% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on cell's survival following exposure to extremely hypoxic conditions, whereas following moderate hypoxia, cells expressing functional p53 enhanced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the induction of apoptosis during hypoxia, other factors seem to be equally important.     

Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.     

The combined use of 131I-labeled antibody and the hypoxic cytotoxin SR 4233 in vitro and in vivo.

Radioimmunotherapy is hindered by the slow penetration of antibody molecules into tumors. Cells that are poorly targeted by antibody, because of their distance from feeding blood vessels, receive the lowest radiation dose, and this problem is compounded if there are radioresistant hypoxic cells present. It would be desirable to combine radioimmunotherapy with an agent that is preferentially toxic to these cells. SR 4233 is a potent hypoxic cytotoxin, and it was combined with 131I-NR-LU-10 to treat LS174T human colon adenocarcinoma multicell spheroids and nude mouse xenografts for these studies. Under conditions of severe hypoxia (< 0.01% O2), 2 h of pretreatment or 18 h of simultaneous treatment with SR 4233 did not significantly enhance the effectiveness of 131I-NR-LU-10 in spheroids. However, under aerobic conditions with a 10% fraction of hypoxic cells, there was more toxicity than would be predicted from simple additivity. Xenografts treated with 131I-NR-LU-10 + SR 4233 had a growth delay that was significantly longer than that achieved with 131I-NR-LU-10 alone. In both spheroids and xenografts, combined treatment produced about 10 times more cell killing than 131I-NR-LU-10 alone. The lack of enhancement in spheroids under complete hypoxia suggests that SR 4233 does not sensitize hypoxic cells to radiation damage. The results with aerobic spheroids and in vivo, where a portion of the cells were hypoxic, could be explained by the targeting of different cell populations (hypoxic and aerobic) by each therapeutic modality. This effect should also be enhanced by reoxygenation and reestablishment of the hypoxic fraction during treatment, thus allowing more than the initially hypoxic fraction of cells to be killed by the SR 4233.     

Hypoxia increases the association of 4E-binding protein 1 with the initiation factor 4E in isolated rat hepatocytes

Incubation of hepatocytes under hypoxia increases binding of translation initiation factor eIF-4E to its inhibitory regulator 4E-BP1, and this correlates with dephosphorylation of 4E-BP1. Rapamycin induced the same effect in aerobic cells but no additive effect was observed when hypoxic cells were treated with rapamycin. This enhanced association of 4E-BP1 with eIF-4E might be mediated by mTOR. Nevertheless, only hypoxia produces a rapid inhibition of protein synthesis. Although hypoxia might be signalling via the rapamycin-sensitive pathway by changing eIF-4E availability, such a pathway is unlikely to be responsible for the depression in overall protein synthesis under hypoxia.     

Calpain is activated during hypoxic myocardial cell injury.

Cell death during hypoxia rose to 80% after 6 h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6 h. This activity was markedly inhibited by calpain-specific inhibitor I (n-acetyl-leucyle-leucyle-norleucinal). beta-Adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. On the other hand, alpha-adrenergic blocking agents did not affect calpain activity and cell death under hypoxic conditions. These results prove that beta-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.     

Buoyant density of EMT6 fibrosarcoma cells

EMT6 fibrosarcoma cells were grown to the exponential phase in tissue culture and incubated at 37°C under hypoxic conditions. Buoyant density was determined as a function of the time in hypoxia. Hypoxia was produced in two ways. The first involved incubation of the cells in sealed aluminum chambers containing 95% N₂, 5% CO₂ gas, and <10 ppm oxygen, resulting in the cells rapidly becoming exposed to the hypoxic environment. After incubation at 37°C, they were centrifuged in linear Ficoll gradients to their isopycnic density. A significant decrease in density was found after 4 h, and prolonged incubation up to 24 h did not result in further change. This density change was reversible on transfer back to aerobic conditions, with the hypoxic cells reverting to their aerobic density after about 10 h reincubation in air. The second method of producing hypoxia involved growing about 8×10⁶ cells in a medium-filled air-tight container. Hypoxia was produced gradually as the oxygen in the medium was consumed by cellular respiration. Similar results were obtained; that is, hypoxic cells became significnatly less dense. However, when the level of hypoxia was varied between 4000 and <10 ppm at 2-h intervals after the cells had depleted all of the original oxygen, no significant difference in density was found between hypoxic and aerobic cells.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

Mechanisms of cell cycle arrest in response to TGF-beta in progestin-dependent and -independent growth of mammary tumors

TGF-beta1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-beta1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G(1) phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-beta1 antiproliferative effects. TGF-beta1 inhibition of cyclins D1 and A expression and up-regulation of p21(CIP1) levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-beta1 inhibition of MPA-dependent and -independent proliferation. TGF-beta1 inhibited cyclin D2 expression and up-regulated p27(KIP1) levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27(KIP1) association with cdk2 in C4HD cells treated with TGF-beta1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-beta-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-beta1 correlated with significantly lower levels of p15(INK4B), p21(CIP1), and p27(KIP1) expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16(INK4). Thus, common targets were found to exist in TGF-beta1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-beta1-inhibited proliferation driven by the progesterone receptor.     

P16Ink4a tumor suppressor function in lung cancer cells involves cyclin-dependent kinase 2 inhibition by Cip/Kip protein redistribution.

As cell cycle regulators whose activity is frequently altered in human cancers, cyclin-dependent kinases (cdks) are novel targets for therapeutic intervention. cdk inhibition is an emerging strategy for the treatment of non-small cell lung carcinomas (NSCLCs) because most derived cell lines express functional retinoblastoma protein (Rb) but appear to bypass its function with inappropriate cdk activity. Elevated cdk4/cdk6 activity in NSCLC cells is often due to inactivation of the p16Ink4a cdk inhibitor. To model the effects of cdk4/cdk6 inhibition, we have expressed p16Ink4a in a Rb-positive NSCLC cell line that lacks endogenous p16Ink4a expression. Whereas cdk4/cdk6 inhibition and Rb dephosphorylation are expected on p16Ink4a expression, we have also observed indirect cdk2 inhibition. cdk2 inactivation by the redistribution of other cdk inhibitors may be required for p16Ink4a-mediated growth suppression of Rb-positive cells. The implications of such a requirement on the use of chemical cdk inhibitors to treat human cancers will be discussed.