Serum deprivation

Burk (1970) showed that Syrian hamster cells (BHK21/C13) failed to grow when transferred to medium containing 0.25% serum and could be maintained in a quiescent state for 8 days or more. On readdition of serum no DNA synthesis occurred for 9 h and mitotic peaks were observed at about 23 and 33 h. It appeared the cells had come to rest in G1 and on stimulation showed a lag of about 9 h before entering into exponential growth with a generation time of about10-12h. 

Adding serum for just 3 h (serum pulse) induces about 50% of the cells to pass through one cycle, and the proportion of committed cells depends almost linearly on the duration of the serum pulse (Brooks, 1976). 

Temin (1970) and Todaro et al. (1965) showed similar effects for chicken fibroblasts and 3T3 mouse fibroblasts. The low level of serum is important for survival as well as for the subsequent stimulation of DNA synthesis (Cherrington, 1984). A kinetic analysis using time lapse cinematography (Zetterberg and Larsson, 1985) showed that Swiss 3T3 cells were only susceptible to cell cycle arrest in a short period (3-4 h) following mitosis. Even a 1-h exposure to serum-free medium during this time forced the cells into GO from which they required 8 h to return to Gl. The length of the postmitotic sensitive phase was very constant at between 3 and 4 h but considerable intercellular variability existed in the duration of the pre S-phase Gl period consistent with a transition probability event ( 10.4). 

This technique is only appropriate for normal cells which on serum deprivation arrest in Gl. Tumour cells often (but not always) arrest in other parts of the cell cycle usually as a result of deficiency in the supply of nutrients rather than as a result of depletion of serum supplied growth factors (Chapter 2). 

To activate confluent or serum-starved fibroblasts, serum is required to provide competence factors such as PDGF or FGF which make cells competent to synthesise DNA, and progression factors such as EGF and IGFs which allow competent cells to progress 

---

Zhang E, Li X, Zhang S et al (2005) Cell cycle synchronization of embryonic stem cells effect of serum deprivation on the differentiation of embryonic bodies in vitro. Biochem Biophys Res Commun 333 1171-1177... 

Goyeneche AA, Harmon JM, Telleria CM (2006) Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis. Reproduction 131 103-111 Gray MW, Burger G, Lang BF (2001) The origin and early evolution of mitochondria. Genome Biol 2 1018.1-1081.5... 

Another important limitation to be aware of is that the induction of apoptosis in a population of cells will not always result in a clearly distinct subdiploid peak (12), We have seen this particularly when apoptosis is induced in Burkitt lymphoma cells by serum deprivation. After approx 2 wk, all cells are apoptotic as can be clearly seen by the two-dimensional light scatter assay and confirmed by microscopic analysis of acridine orange-stained cells, but the cell-cycle profile gives the appearance of a mainly viable population (Fig. 5). 

Fig. 5. Comparison of two-dimensional light scatter assay with subdiploid DNA peak assay. Burkitt lymphoma cells were serum-deprived for 14 d and then assayed for (A) forward light scatter (FSC) versus 90° light scatter (SSC) or (B) cell cycle. Note that although all the cells are clearly apoptotic as shown by the light scatter assay, there is only a small subdiploid peak (SD) present in the cell-cycle analysis, demonstrating that in this case, celt-cycle analysis alone would grossly underestimate the extent of apoptosis present in the cell population.

Gasull T., DeGregorio-Rocasolano N., Zapata A., and Trullas R. (2000). Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation. J. Biol. Chem. 275 18350-18357. 

Selegiline delays apoptosis in serum-deprived PC12 cells. 

Hogg N, Browning J, Howard T, Winterford C, Fitzpatrick D, Gobe G. Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and transforming growth factor-beta. Endothelium 1999 7 35-39. 

GM1 prevented apoptotic cell death by enhancing TrkA dimerization and consequent autophosphorylation in PC 12 cells (Ferrari and Greene, 1998) and decreased the severity of ischemic brain lesions in experimental models (Frontczak-Baniewicz et al., 2000 Hicks et al., 1998). Also in neuron-rich cortical cultures, GM1 and other gangliosides attenuated serum deprivation-induced neuronal apoptosis (Ryu et al., 1999). 

BEO to restore Akt and GSK-3/3 phosphorylation reduced by serum deprivation (Corasaniti et al., 2007). Collectively, these observations indicate that a PI3-K-dependent mechanism mediates the effects of BEO on Akt phosphorylation and this may conceivably account for the present in vivo observation. 

Recently, Mu et al. (2003) have reported that the KCNK9 potassium channel (TASK) gene is amplified and overexpressed in breast cancers, lung and prostate cancers. Moreover, overexpression of KCNK9 was found in 57 (46.0%) of the 124 patients with colorectal carcinomas, but not in the patients with colorectal adenoma (Kim et al. 2004). Interestingly, overexpression of KCNK9 promotes tumor formation and induces resistance to both hypoxia and serum deprivation. 

Wu et al. (1998) noted that doxorubicin-induced apoptosis in lymphoid cells was blocked by pepstatin A, which is an inhibitor of cathepsin D. These investigators also observed that cathepsin D was induced through p53 DNA-binding sites at the cathepsin D promoter. Moreover, they have found that, compared to fibroblasts from wild-type mice, cathepsin D-/- fibroblasts from gene knock-out mice exhibited increased resistance to death caused by doxorubicin. Also, in serum-deprived rat PC 12 cells undergoing apoptosis, the amount of cathepsin B has been observed to decline, while the level of cathepsin D increased (Shibata et al, 1998), and, in our laboratory (Kagedal et al., 2001), the same phenomenon was recently seen in human fibroblasts exposed to naphthazarin. 

Sphingosine 1 -phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production, J. Biol. Chem. 276 10627-10633. 

As already mentioned (section 2.2) serum is a source of growth factors, so it is perhaps not surprising that the withdrawal of serum from the growth media of most cultured mammalian cells results in notably reduced rates of cell proliferation [18]. Another readily observed feature of serum deprivation is an increase in the level of cellular lipid peroxidation [39], In part this may be a function of the absence of serum components that might provide an antioxidant function, e.g. a-tocopherol, ascorbate, urate, caeruloplasmin, etc. (see also... 

Caspase-2 lch-1 (human), Nedd2 (rat, mouse) Golgin-160, Lamins ( ) Apoptosis (activity suppressed by serum deprivation)... 

Xie L, Li W, Winters A, Yuan F, Jin K, Yang S (2013) Methylene blue induces macroau-tophagy through 5 adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation. Front Cell Neurosci 7 56... 

---