Housekeeping Genes in Cancer

SHORT TECHNICAL REPORTS genes has deviated from the norm. Housekeeping genes in cancer: normalization Overexpression of ribosomal proteins of array data has been reported in certain cancers: colorectal (12), liver (13), and breast Anis H. Khimani1, Abner M. Mhashilkar2, Alvydas Mikulskis1, Michael (14). Recently, overexpression of O’Malley1, Jennifer Liao2, Eva E. Golenko1, Pat Mayer1, Sunil Chada2, ribosomal proteins L7a and L37 has Jeffrey B. Killian1, and Steven T. Lott1 been reported in prostate cancer tissues 1 2 when compared to a normal prostate PerkinElmer Life Sciences, Boston, MA and Introgen Therapeutics, Houston, TX, USA epithelial cell line (15). An examination of the expression of 15 different house- BioTechniques 38:739-745 (May 2005) keeping genes in colon cancer demon- strated substantial changes, particu- Biological maintenance of cells under variable conditions should affect gene expression larly in those coding for metabolic of only certain genes while leaving the rest unchanged. The latter, termed “housekeeping enzymes (16). Interestingly, this study genes,” by definition must reflect no change in their expression levels during cell develop- found little difference in ribosomal ment, treatment, or disease state anomalies. However, deviations from this rule have been proteins. In sharp contrast, exami- observed. Using DNA microarray technology, we report here variations in expression lev- nation of melanocytic lesions showed els of certain housekeeping genes in prostate cancer and a colorectal cancer gene therapy minimal variation between nevi and model system. To highlight, differential expression was observed for ribosomal protein genes melanoma (17). Housekeeping gene in the prostate cancer cells and β-actin in treated colorectal cells. High-throughput differen- variation has also been observed in tial gene expression analysis via microarray technology and quantitative PCR has become normal, primary cell cultures. Savonet a common platform for classifying variations in similar types of cancers, response to che- et al. (18) examined the expression of motherapy, identifying disease markers, etc. Therefore, normalization of the system based three common housekeeping genes on housekeeping genes, such as those reported here in cancer, must be approached with (glyceraldehyde-3-phosphate-dehydro- caution. genase, β-actin, and cyclophilin) in primary cultures of normal thyrocytes under different mitogenic stimulations using Northern blot analysis. As in the INTRODUCTION Normalization of microarray data previous examples, the vast majority involves standardizing the data against of published observations addressing Microarray technology has revolu- a set of reference points between the housekeeping gene-based normal- tionized the era of functional genomics two comparative mRNA populations. ization consist of single gene or small by enabling a global screening of One method to perform this normal- gene sets offering limited usefulness differential gene expression between ization in any differential expression outside of the model system under comparative biological samples. It has methodology is to use housekeeping study (19–23). provided the momentum to keep pace genes as reference standards. House- Normalization issues for large-scale with the rapidly emerging sequence keeping genes, also termed “mainte- gene expression studies performed on information from the human genome nance genes,” by definition maintain microarrays have, for the most part, project. The technology has evolved the basic metabolic functions of the moved away from simple comparison from the basic probe-to-target, gene- cell and provide support through the to a set of housekeeping genes to by-gene hybridization on a Northern cell cycle and, thus, are expected to more complex statistical analyses that blot (1,2) and from a grid screening of remain unchanged in their expression account for overall expression levels cDNA libraries (3) to multiple probe levels through various tissues or cells. of all genes (24–29). Although these to multiple target hybridizations of up However, with increasing expression methods are quite effective when to two comparative samples for tens studies using high-throughput technol- dealing with extremely large data sets, of thousands of genes (4). This rapidly ogies, the general concept of the laboratories examining a small set of evolving technology has proved to be a constant expression of housekeeping genes by microarray hybridization or powerful tool in global gene expression genes is controversial, and their use as alternative technologies (e.g., real-time analyses in various organisms (5,6), references is being approached with PCR, Northern blot analysis, etc.) are drug target validation (7), and identi- caution. Although a set of hundreds of left with little guidance in selecting fication of disease-specific genes and these genes has been designated to be appropriate reference genes. diagnostics (8). Recent advances in continually expressed through human To illustrate this dilemma, we have cancer research using microarrays development (11), the reliability of performed differential gene expression have been made by the identification these genes as internal standards studies using microarray technology of distinct forms of large B-cell in gene expression experiments, on three model systems: the prostate lymphomas (9) and by the distinction following differential treatments or cancer cell lines, LNCaP and PC3, between acute myeloid leukemia and during diseased states, is precarious. a colorectal cancer gene therapy acute lymphoblastic leukemia, with a One such example is cancer, in which model, and a phorbol ester [phorbol- direct impact on therapy (10). the expression of some housekeeping 12-myristate-13-acetate (PMA)]- Vol. 38, No. 5 (2005) BioTechniques 739 SHORT TECHNICAL REPORTS Table 1. Samples, Labeling Methodology, and Microarrays Used for These Experiments as controls for comparison of differ- Sample Name and Label Labeling Method Microarray Hybridized ential gene expression. Total RNA was isolated using TRIzol® (Invit- Cy3 Cy5 Direct TSA 2400 Gene Array Cancer Array rogen) and RNeasy® RNA isolation kit PC3 LNCaP X X X X columns (Qiagen, Valencia, CA, USA), Jurkat PMA-Stimulated Jurkat X X X as described by the manufacturers and by a combined protocol as described DLD-1 Ad-MDA7-DLD-1 X X in the MICROMAX™ Human cDNA TSA, tyramide signal amplification; PMA, phorbol-12-myristate-13-acetate. System I-Direct manual (PerkinElmer Life Sciences, Boston, MA, USA). stimulated Jurkat T-cell line model. In from the ATCC (Rockville, MD, USA). contrast to prior studies, we detail the All three cell lines were propagated in Adenoviral Vector Transduction expession levels of 42 common house- RPMI 1640 (Invitrogen, Carlsbad, CA, keeping genes. These data illustrate the USA) with 10% fetal bovine serum Adenovirus-MDA7 (Ad-MDA7) need for the careful selection of appro- (FBS). DLD-1, a colorectal cell line, was constructed at Introgen Thera- priate housekeeping genes for smaller and T47D, a ductal carcinoma of breast peutics (Houston, TX, USA). Adeno- scale gene expression experiments. cell line, were also obtained from the virus-luciferase (Ad-luc) was also ATCC (nos. CCL-221 and HTB-133, constructed at Introgen Therapeutics respectively). Both cell lines were and was used in control vector experi- MATERIALS AND METHODS propagated in Dulbecco’s modified ments (data not shown). Cancer cells Eagle’s medium (DMEM; Invitrogen) were plated at 5 × 105 per well in a 6- Cell Culture and RNA Isolation with 10% FBS. Jurkat cells (human well format; 24 h later, the cells were leukemic T-cell line) were stimu- infected at a multiplicity of infection Prostate cancer cell lines, LNCaP lated with PMA at 50 ng/mL for 4 h. (MOI) of 1000 viral particles per cell and PC3, and Jurkat cells were obtained Unstimulated Jurkat cells were used for 3 h. After infection, the cells were washed with DMEM and further cells was labeled via cDNA synthesis incubated in DMEM at 37°C in a 10% and co-hybridized on the microarrays as CO2 incubator (Steri-Cult 200; Forma described above. In a third set of differ- Scientific, Woburn, MA, USA). At ential gene expression analysis experi- different time intervals [24, 48, 72, ments, total RNA from Ad-MDA7- and 96 h post-infection (p.i.)], the cells transduced colorectal cancer cell line, were trypsinized and lysed with sample DLD-1, was labeled with Cy5-dUTP, buffer used for sodium dodecyl sulfate and the comparative RNA from control polyacrylamide gel electrophoresis vector-transduced cells was labeled with (SDS-PAGE). The lysate was vortex Cy3-dUTP. Respective pairs of labeled mixed briefly, boiled at 100°C for 2 targets were co-hybridized on the min, centrifuged (at 15,300× g for 2 human cancer gene microarray. Prior to min), run on SDS-PAGE, and Western hybridization, the probe, resuspended blotted. The blot was probed for β- in 20 µL of hybridization buffer, was actin monoclonal antibody (Sigma, St. denatured at 90°C for 2 min, placed Louis, MO, USA). on ice, centrifuged briefly, and applied to the microarrays with a coverslip in Differential Gene Expression- place. Hybridizations were performed Microarray Analysis at 65°C overnight in the hybridization buffer provided in the MICROMAX The following systems were used to kits. Following hybridization, strin- perform the differential gene expression gency washes were performed as analysis: (i) the MICROMAX Human described in the kit protocol. In addition,

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