Topic Article:
Gadd45a Activation Protects Melanoma Cells from Ultraviolet B-Induced Apoptosis
Caroline Fayolle, Julie Pourchet, Claude Caron de Fromentel, Alain Puisieux, Jean-François Doré and Thibault Voeltzel
Journal of Investigative Dermatology (2008) 128, 196–202; doi:10.1038/sj.jid.5700963

The Role of Gadd45a in the Survival of Melanocytes and Melanoma Cells

Vidya Rajpara¹, Shasa Hu¹ and Robert S. Kirsner¹

Journal of Investigative Dermatology (2008) 128, 4; doi:10.1038/sj.jid.5701198

Despite improvements in prevention and screening, mortality from advanced melanoma has not improved (Geller et al., 2007) and ultraviolet radiation (UV) is a major risk factor for melanoma development (Fears et al., 2002). Greater understanding of the mechanisms of melanoma development may lead to novel treatment options. As opposed to keratinocytes, which routinely undergo apoptosis after UVB irradiation, melanocytes resist apoptosis. In previous work, melanocytes and melanoma cells responded to UVB by activating an original pathway involving Gadd45a, one of the several growth-arrest and DNA damage–inducible genes (Lefort et al., 2001).

Fayolle et al. (2008) describe in greater detail the relationship between melanoma cells and UVB-induced apoptosis mediated by Gadd45a, an apoptosis regulator gene. After production of Gadd45a was inhibited, the authors found that melanoma cells reduced colony formation and underwent cell death after UV irradiation, unlike cells with uninhibited Gadd45a (Fayolle et al., 2008). To better understand these findings, the investigators explored the mechanisms of this UVB-induced cell death and determined that it was due to apoptosis rather than mitotic catastrophe. They demonstrated that inhibition of Gadd45a led to increased levels of pro-apoptotic proteins (caspase-3) and decreased levels of anti-apoptotic proteins (Bcl-xL) after UVB exposure, leading to melanoma cell death.

Through the following questions, we will examine this paper in greater detail.

QUESTIONS

1. What are the known functions of Gadd45a, and how was it hypothesized to be important in melanoma pathogenesis?

2. What is shRNA, and why was it important for this study?

3. Why was it important for the authors to differentiate apoptosis from mitotic catastrophe as a response to UVB in melanoma cell lines? What surrogate markers of apoptosis did the authors use in their experiments?

4. What can be concluded about the role of Gadd45a in melanoma survival based on this article?

5. What may be the clinical implications of this article?

ANSWERS

1. Gadd45a is a protein involved in DNA damage responses (Gadd = growth arrest and DNA damage–inducible genes). In differing cell types, Gadd45a appears to function differently; its known functions include cell-cycle arrest at G2 and an association with apoptosis, DNA repair, and genomic stability. The role of Gadd45a in apoptosis is controversial, as some studies have found that it promotes apoptosis (Takekawa and Saito, 1998), while others report that Gadd45a protects cells from apoptosis and promotes cell survival (Smith et al., 1996). Gadd45a interacts with several other apoptosis regulator proteins, and some of these mechanisms function independently of p53 (Lieberman and Hoffman, 2007). Gadd45a may regulate apoptosis, arresting the G2 cell cycle to allow for either DNA repair or apoptosis, depending on what other regulator proteins are induced.

Melanocytes and melanoma cells respond to ultraviolet light B (UVB) by activating Gadd45A expression in a pathway independent of p53 (Lefort et al., 2001); Gadd45a expression is upregulated when these cells are exposed to UVB, which in turn prevents cells from undergoing apoptosis. When Gadd45a expression is blocked, melanoma cells lose this protection and experience cell death via apoptosis.

2. Small-hairpin RNAs are used to silence gene expression. They are called “hairpin” because the complementary RNA sequence at one end causes a loop. By using a plasmid vector, shRNA can be introduced into cells and be continuously expressed as a result of its promoter sequence. In this study, the investigators used shRNA to target the Gadd45a mRNA sequence. Two different shRNAs were used to inhibit the expression of Gadd45a. Cell proliferation and apoptosis of melanoma cells treated with these shRNAs were compared to those of control cells following UVB therapy and chemotherapy. By negating the mediating effects of Gadd45a, the investigators demonstrated that it plays a significant role in regulating melanocyte and melanoma cell apoptosis following UVB irradiation. For example, when Gadd45a activation was inhibited, the melanoma cells grew less well, and Gadd45a activation was also inhibited by exposure to UVB. Finally, the survival of these cells after UVB exposure was dependent on the expression of Gadd45a.

3. The investigators demonstrated that UVB induces apoptosis rather than mitotic catastrophe of melanoma cells. Mechanisms by which UVB can induce apoptosis include the mitochondrial pathway, death receptor pathway, and apoptosis as a result of exposure to reactive oxygen species (Kulms et al., 2002). Previous studies have shown that after UVB irradiation, melanoma cells accumulate in the G2 phase, and Gadd45a-deficient cells lack this important checkpoint (Fayolle et al., 2006). By comparing the behavior of melanoma cells expressing Gadd45a and melanoma cells in which Gadd45a expression is impaired following exposure to either UVB or a chemotherapeutic agent (paclitaxel) known to interfere with mitosis and cause mitotic catastrophe, the mechanism by which Gadd45a and UVB affect cell growth could be studied. Compared with paclitaxel, UVB did not cause a mitotic catastrophe but, rather, induced apoptosis.

After UVB treatment, melanoma cells lacking Gadd45a had lower levels of pro-caspase-3 and increased caspase-3 activity, both of which are markers of apoptosis. In addition, these cells exhibited decreased levels of Bcl-xL, an anti-apoptotic protein that counterregulates Bcl-2 and other pro-apoptotic proteins. By demonstrating the relation between Gadd45a and these pro- and anti-apoptotic proteins without any evidence of mitotic catastrophe, the authors concluded that UVB induces apoptosis in Gadd45a-deficient melanoma cells. These apoptosis pathways were independent of p53 (Fayolle et al., 2007), which is significant as Gadd45a interacts with many other apoptosis-regulating genes. For example, in cell lines where Gadd45a expression was inhibited, the investigators did not find a complete reduction in cell growth, nor did all cells undergo apoptosis. Additionally, inhibition of Gadd45a expression made melanoma cell lines more sensitive to several chemotherapeutic agents such as cisplatin and dacarbazine.

4. This study included several findings. In melanoma cell lines in which shRNA inhibited Gadd45a expression, there was decreased cell survival compared with controls. These same cell lines also had diminished Gadd45a activation after UVB exposure.

Based on these results, it appears that Gadd45a protects melanoma cells from apoptosis, thereby enhancing their survival. Gadd45a may participate in the maintenance of Bcl-xL concentration regulating pro-apoptotic proteins in the Bcl-2 family. Gadd45a may be an important gene for melanoma survival, in part by inhibiting apoptosis, but as cell survival and death result from multiple interacting genes, Gadd45a may act at different points in the cell cycle, and with significant overlap. Gadd45a may also protect melanoma cells from chemotherapy.

5. While more information is needed about the Gadd45a gene’s function on melanoma cells, it appears to be important in cell regulation, especially after oncogenic stimuli such as UVB irradiation. However, the activity of Gadd45a is not specific to melanoma cells; it appears to have functions—in some cases, similar functions—in other cell types, such as myeloid cells (Lieberman and Hoffman, 2007). Using this information to envision potential treatments, an agent that could selectively inhibit Gadd45a in melanoma cells could be used as an adjunctive therapy to enhance the efficacy of chemotherapy, similar to the effects demonstrated in vitro in this study. In addition, Gadd45a may be a useful gene in identifying people at risk for melanoma. For example, there may be a subset of patients that have differing amounts or responsiveness of Gadd45a activation from other patients or from the general population. This subset may therefore prohibit their abnormal melanocytes from undergoing apoptosis, and be at higher risk of malignant transformation and/or proliferation.

REFERENCES

Fayolle C, Pourchet J, Cohen A, Pedeux R, Puiseuix A, de Fromentel C et al. (2006) UVB-induced G2 arrest of human melanocytes involves Cdc2 sequestration by Gadd45a in nuclear speckles. Cell Cycle 5:1859–64

Fayolle C, Pourchet J, Caron de Fromentel C, Puisieux A, Doré J-F, Voeltzel T (2008) Gadd45a activation protects melanoma cells from ultraviolet B–induced apoptosis. J Invest Dermatol 128:196–202.

Fears TR, Bird CC, Guerry D, Sagebiel RW, Gail MH, Elder DE et al. (2002) Average midrange ultraviolet radiation flux and time outdoors predict melanoma risk. Cancer Res 62:3992–6

Geller AC, Swetter SM, Brooks K, Demierre MF, Yaroch AL (2007) Screening, early detection, and trends for melanoma: current status (2000–2006) and future directions. J Am Acad Dermatol 57:555–72

Kulms D, Zeise E, Poppelmann B, Schwartz T (2002) DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way. Oncogene 21:5844–51

Lefort K, Rouault JP, Tonderaeu L, Magaud JP, Doré JF (2001) The specific activation of gadd45 following UVB radiation requires the POU family gene product N-oct3 in human melanoma cells. Oncogene 20:7375–85

Lieberman DA, Hoffman B (2007) Gadd45a in the response of hematopoietic cells to genotoxic stress. Blood Cells Mol Dis 39:329–35

Smith ML, Kontny HU, Zhan Q, Sreenath A, O’Conner PM, Fornace AJ Jr. (1996) Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene 13:2255–63

Takekawa M, Saito H (1998) A family of stress-inducible GADD45-like proteins mediate activation of the stress responsive MTK1/MEKK4 MAPKKK. Cell 269:64–72

¹Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA