Loss of protein kinase C delta gene expression in human squamous cell carcinomas: a laser capture microdissection study

Abstract

Protein kinase C delta (PKC-delta) protein levels are frequently low in chemically and UV-induced mouse skin tumors as well as in human cutaneous squamous cell carcinomas (SCCs). Furthermore, overexpression of PKC-delta in human SCC lines and mouse epidermis is sufficient to induce apoptosis and suppress tumorigenicity, making PKC-delta a potential tumor suppressor gene for SCCs. Here we report that PKC-delta is lost in human SCCs at the transcriptional level. We used laser capture microdissection to isolate cells from three normal human epidermis and 14 human SCCs with low PKC-delta protein. Analysis by quantitative reverse transcription-PCR revealed that PKC-delta RNA was reduced an average of 90% in the SCCs tested, consistent with PKC-delta down-regulation at the protein level. Analysis of DNA from nine of the same tumors revealed that PKC-delta gene was deleted in only one tumor. In addition, Ras-transformed human keratinocytes, which have selective down-regulation of PKC-delta at both protein and mRNA levels, had significantly repressed human PKC-delta promoter activity. Together, these results indicate that PKC-delta gene expression is suppressed in human SCCs, probably via transcription repression. Our results have implications for the development of topical therapeutic strategies to trigger the re-expression of pro-apoptotic PKC-delta to induce apoptosis in SCCs.

Figure 1

PKC-δ immunostaining in SCCs and LCM. A: Loss of PKC-δ protein in human SCCs. Hematoxylin and eosin staining of normal human skin (NN363) and human SCC (case 234) is shown on top. PKC-δ staining of normal human epidermis and human skin SCCs by immunohistochemistry is shown in the middle row. Keratin-14 staining of sections of normal human epidermis and human skin SCCs is shown at the bottom. Keratin-14 staining was used as an epithelial marker to differentiate between cells of epithelial and non-epithelial origin. Scale bars = 30 μm. B: Isolation of pure SCC and normal epidermis cells by LCM. The tissue before and after LCM are shown. The isolated region containing normal epidermal cells and SCC cells on the cap are shown. C: Detection of PKC-δ RNA. RNA was purified from normal human epidermis isolated by LCM. The integrity and specificity of the PKC-δ RNA sample was verified by performing RT-PCR in presence and absence of reverse-transcriptase as shown. The predicted 260 bp PCR fragment was obtained on performing PKC-δ RT-PCR in the presence of reverse transcriptase.

Figure 2

Quantification of PKC-δ mRNA and gene in human SCCs. A: PKC-δ RNA is reduced in human SCC cells compared with normal human epidermis. Keratinocytes from 14 human SCC samples and 3 normal epidermises were isolated by LCM and analyzed for PKC-δ RNA by qRT-PCR. The PKC-δ RNA levels were normalized to the GAPDH RNA for each sample. The sample numbers are shown on the x axis, and the relative PKC-δ mRNA levels above each bar. B: PCR-based gene deletion assay was performed on cells isolated by LCM from 9 human SCCs and 4 normal human epidermises. PKC-δ gene fragment was detected by specific primers for a 100 bp region inside intron 1. GAPDH gene fragment was amplified to normalize for DNA integrity and amount. The sample numbers are shown on the x axis, and the relative PKC-δ gene levels are indicated above each bar. Error bars denote SD for 3–4 independent qPCR reactions. Products from a representative qPCR reaction for PKC-δ and GAPDH were run on agarose gels and are shown below the bar graph. Note that only one SCC (205) had a deletion in the PKC-δ gene.

Figure 3

PKC-δ gene expression is reduced in Ras transformed keratinocytes. A: PKC-δ RNA is reduced in Ras transformed HaCaT cells. Total RNA was isolated from HaCaT, HaCaT Ras I-7 and HaCaT Ras II-4 cells and analyzed for PKC-δ RNA by qRT-PCR. B: PKC-δ promoter activity is down-regulated in Ras transformed HaCaT cells. pGL3-Basic or pGL3-hPKCδ−4.4 was transiently co-transfected with pRL-TK into HaCaT and HaCaT-Ras cells. After 48 hours, luciferase activity in cell lysates was measured. Shown is the mean and SD from experiments performed in triplicate. *P < 0.001.