The UOK 257 Cell Line – A Novel Model for Studies of the Human Birt-Hogg-Dubé Gene Pathway^***

2.1 Case Report

The patient was a previously healthy 46 year old male who initially presented with acute onset gross hematuria. His past medical history was significant for history of two previous episodes spontaneous pneumothorax and the diagnosis of numerous fibrofolliculomas on the scalp, face, neck and trunk. During his workup for hematuria, the patient underwent computed tomography (CT) scan chest, abdomen, and pelvis revealing a 7 × 5 cm multi-nodular enhancing mass encompassing the upper pole of the left kidney, no solid lesions in the right kidney, and multiple pulmonary cysts. Otherwise, CT scan revealed no evidence of disease in the remainder of the chest, abdomen and retroperitoneum. Given the presence of both the dermatologic findings and the renal mass, the patient underwent genetic testing and was found to have a germline mutation in the FLCN gene and a diagnosis of BHD syndrome was made. Following diagnosis and evaluation, the patient underwent an open left radical nephrectomy via an 11^th rib flank incision. Pathology revealed clear cell renal cell carcinoma (clear cell RCC), Fuhrman grade 3 of 4, with evidence of invasion into the hilar adipose tissue. Multiple hilar lymph nodes, removed at the time of resection, revealed no evidence of tumor. Furthermore, although the mass was predominantly clear cell RCC, the pathologist noted foci of tubular papillary and chromophobe histologies. Approximately eight months following surgery, the patient presented with a four day history of abdominal discomfort and diarrhea. He underwent a CT scan of the abdomen/pelvis, which revealed a 3 cm soft tissue mass in the left renal fossa. He subsequently underwent a percutaneous CT guided biopsy of the retroperitoneal mass, which revealed recurrent renal cell carcinoma that was morphologically consistent with the patient’s previous RCC tumor. Further metastatic imaging including a whole-body bone scan, MRI head, and PET scan, revealed foci of metastatic disease in the left renal fossa, left iliac crest, and left lesser trochanter. He subsequently underwent several cycles of systemic therapy including high dose interleukin 2 (IL 2), interferon, and an investigational agent (phase 1 clinical trial). The disease continued to progress through the therapies, was eventually was placed on palliative care measures, and, approximately 21 months after initial diagnosis and 13 months following the diagnosis of metastatic disease, he died of progressive metastatic renal cancer.

2.2 Tumor Origin and Pathologic Analysis

The surgical specimen from a left nephrectomy weighted 1,213.2 grams, consisting of a kidney and adrenal gland with overlying fascia and adipose tissue. The tumor mass measured approximately 8.0 × 8.0 × 8.0 cm and had completely distorted in the upper pole of kidney. The multiple nodules of friable tumor mass were present primarily in the superior pole of the kidney and were invading the collecting system. Further dissection revealed a yellow/tan homogeneous tumor mass within the nodule. A portion of the tumor was retained in the laboratory for viable in vitro culture. Portions were submitted for electron microscopy, and the remainder was retained for permanent histopathological diagnosis. Prior to surgery, the patient was consented on an internal review board approved tissue procurement protocol.

2.3 Tissue Culture, Establishment of Transplantable Cell Line and sub-clone lines

A portion of tissue was removed from the middle of the tumor mass approximately 10-min after nephrectomy and tissue extraction. Using sterile technique, the tissue was immersed into a small aliquot of 1x PBS buffer, minced with a sharp scalpel to obtain approximately 1 mm diameter fragments, and then subsequently plated onto a 60mm Petri dish. Dulbecco’s modified Eagle’s medium (DMEM) containing high glucose (4.5g/L), L-glutamine (2 mM), supplemented with penicillin (50 units/ml), streptomycin (100µg/ml), and MEM non essential amino acids (1x, from 100x solution. Invitrogen, MD), with 10% of heat-inactivated FBS was slowly added to the Petri dish. The volume of medium was adjusted so that there was enough media to cover the tissue fragments, but not enough to cause them to float. The culture was then incubated at 37°C in a humidified atmosphere containing 5% CO₂. Approximately 36 hours after plating, adherent epithelial cell outgrowth from tissue debris was observed. An additional 1–2 ml of fresh DMEM was then added. The first passage was carried out three to five days after plating by a light treatment with 0.05% trypsin-EDTA, while monitoring the cells under an inverted tissue culture microscope. Subsequent passages were carried out every 3–5 days in same manner; the line was propagated for over 50 passages. The established primary line was designated UOK 257. From the parental stock of passage 6, limiting dilutions were performed, and resulting sub-clones were isolated and cultured for more than 15 passages. The sub-clones were numbered, following the name of the primary line (e.g. UOK 257 SC6 refers to subclone 6 of the primary cell line UOK 257).

Approximately 3 ×10⁶ cells in 1x PBS 0.2ml each volume was subcutaneously injected into SCID/BEIG mice to determine the tumorigenic potential of the cells. Xenograft tumor specimens were reimplanted into new mice for multiple generations, and xenografts from the mice were also cultured in the laboratory. Animal care was approved and was in accordance with the guidelines of the National Cancer Institute.

2.4 Cell Proliferation and Metaphase Preparation

When the UOK 257 line culture reached the proliferation phase (i.e. seeded at ~40% confluence and grown and harvested on third day at ~ 90% confluence), the proliferative activity and cellular kinetics were analyzed using automated flow-through cytometer (BD FACScalibur E1806, BD Biosciences, San Jose, CA). The protocol and technical control used was as previously described[13] with some modifications. Briefly, the cells (10⁶/ml) were washed with PBS and fixed in 70% ethanol for at least 1 hour, or the cells were incubated at 4 ° C overnight, washed twice with PBS, and incubated with 50 µl of RNase in 1 mL PBS plus 0.1% bovine serum albumin for 30 minutes. A DNA histogram was obtained by staining cells with 7-Aminoactinomycin D (5 µl (0.25 µg) / 10⁶ cells) (BD Biosciences Pharmingen) and incubating 10 minutes before analysis. Data was analyzed using FlowJo version 6.3.2 software (Tree Star, OR).

At passage 18, UOK 257 cells were treated with Colcemid (10 µl/ml, Boehringer Mannheim [Roche Diagnostics Corporation, USA]) for 1 hr, subjected to hypotonic treatment (0.075 M KCl) and fixative (methanol-acetic acid, 3:1) as described[12]. Chromosome spreads were prepared in a humidity chamber (Thermotron, Model CDS-5, Holland, MI). Metaphase chromosome preparation was as previously described[14]. See: http://www.nature.com/nprot/journal/v1/n6/abs/nprot.2006.358.html]

2.5 Cell Pellet Fixation and Thin Section for Ultrastructural Studies

Cell monolayers were washed with PBS, scraped and fixed in 2.5% PBS-buffered glutaraldehyde, post-fixed in osmium tetroxide, dehydrated and embedded in Spurr’s epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed in a Philips CM10 transmission electron microscope (Philips Electronics, Mahway, NJ).

2.6 DNA Probes Preparation and DNA Sequencing Analysis

BAC based DNA hybridization probes for FISH were prepared by using Vysis Nick-translation Kit (Abbott Molecular Inc./Vysis, IL) incorporated with Spectrum-Orange or Spectrum-Green. Sky painting probes were prepared in-house, as detailed in: http://www.riedlab.nci.nih.gov/protocols.asp. Germline mutation sequencing analysis for the cell line was performed on our lab ABI PRISM™ 310 Genetic Analyzer (Perkin Elmer, CA).

2.7 Sky Analysis and FISH with Chromosome-specific Probes

Fifteen UOK 257 metaphase spreads were analyzed and scored for the following: chromosome number (ploidy), numerical and structural aberrations. Spectrum-based classification and analysis of the fluorescent images (SKY) was achieved using SkyView™ software (Applied Spectral Imaging, Vista, CA). The chromosome complements (karyotypes) of every metaphase spread were analyzed using human chromosome nomenclature rules described in ISCN, 2005[15]. FISH analysis using a chromosome painting probe for chromosome 8, and a gene loci probe for MYC (8 q24.3) (Abbott Molecular Inc./Vysis, IL) was used to assess the copy number of this oncogene and to better define aberrant chromosomes containing small portions of chromosome 8.

Structural aberrations are considered clonal if two or more cells contain the same aberration[15]. With respect to numerical aberrations, gains of chromosomes are considered clonal if two or more cells contain the same extra chromosome, and losses are classified as clonal if they are observed in three or more cells[15].

The karyotype for UOK 257 is shown in Figure 4 and [has been entered into the NCBI, SKY/CGH interactive online database; http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi (submitter: Padilla-Nash)(public access pending publication). In this database, the patient’s karyotypes are depicted as a colored human ideogram, called a SKYGRAM. Each SKYGRAM presents the complement of normal and abnormal chromosomes with the position of their breakpoints linked to the NCBI DNA sequence database.]

2.8 Image Acquisition

The UOK 257 cell morphology was examined with an inverted microscope with a digital camera [Olympus 1X71, Olympus Optical Co. LTD. Japan]. Images were acquired with a 10x objective from 6–8 random fields per culture. The cell counting and mean cell size (micron) were analyzed with Cellometer Auto T4 image-based cell counter. (Nexcelom Bioscience, Lawrence, MA). The ultrastructural images on thin section were taken on a ZEISS EM109 electronic microscope (at x6500, x11,000 and high magnification x21,000). FISH fluorescence images were acquired by Leica DMR XA [Leica Microsystems, GmbH], with loaded IP lab version 3.7 software, plus customized scripts. Image acquisition of SKY-labeled metaphase chromosome spreads were carried out by using a SpectraCube™ (Applied Spectral Imaging, Vista, CA) and a charge-coupled device camera on top of an epifluorescence microscope equipped with custom-designed-triple band pass optical SKY filter (Chroma Technology, Rockingham, VT). Analysis of the metaphase spread karyotypes was performed using SkyView™ software (ASI)[12].

3.1 Histopathology

Fig. 1 (A–D) shows the light microscope appearance of an aggressive renal cell carcinoma derived from this individual surgical specimen. The tumor was characterized as being predominantly atypical clear epithelial cell type (Fig. 1A), with enlarged, round to irregular nuclei, prominent nucleoli and moderate to abundant vacuolated cytoplasm seen as single cells, clusters, and sheets. The tumor also demonstrated a variety of histologies with detailed junctional complexity including tubular papillaries (Fig. 1B), solid areas (Fig. 1C), and notably, foci reminiscent of chromophobe renal cell carcinoma (Fig. 1D). Ten SKID/BEIG mice injected with the UOK 257 primary cell line showed outgrowth within three weeks post injection, and palpable tumors were formed in 10 out of 10 mice. Twenty-two weeks after injection, tumor size was measured at 1.5 cm in diameter. H & E slides were processed from tumor xenografts, revealing pathologic features similar to that of the case report. Notably, in some of the slower growing tumors aged 28 weeks in mice by the time of euthanization, pathologic analysis showed features not only of the predominantly clear cell type (Fig. 2E), but also complex junctions with papillary architecture, solid areas of cells with eosinophilic cytoplasm lined by clear cells (Fig. 2H), and chromophobe foci surrounding the clear cell tumor area (Fig. 2G). Concurrently, xenograft tumor specimens were reimplanted into new mice for several generations. They were also brought back to lab for proliferation cultures, and retained the ability to grow in culture. A subline derived from the xenograft, designated UOK 257 XRP1., when injected back into SKID/BEIG mice, resulted in a more rapid outgrowth than the parent line.

3.2 Cell proliferation, cell cycle distribution and ploidy pattern

Morphological aspects of primary human hereditary renal carcinoma cultures are shown in Fig. 2 B. The UOK 257 cells cultured in vitro show an adherent monolayer cultured epithelial cell phenotype and display a loss of contact inhibition. Unlike other renal cell lines, the UOK 257 cells form into small patches of cellular islands in culture at ~50% confluence. Cell cycle and ploidy pattern of UOK 257 cells were evaluated by flow cytometric DNA histogram of 7-AAD-stained tumor cells, which were cultured three days to reach around 90% confluence. The distribution of the tumor cell population at each phase in the cell cycle is given in Fig. 2C.

3.3 Ultrastructural analysis

The cells exhibited ultrastructural features of tubular epithelial cells, such as microvilli on one (apical) surface and basal lamina on the opposite surface (Fig. 3A). Basal lamina-like material was focally abundant in-between tumor cells (Fig. 3B). Desmosomes and tight junctions were present. Variable numbers of mitochondria were seen in the tumor cells. In some of them the mitochondria were abundant consistent with oncocytic change (Fig. 3C). Cytoplasmic microvesicles similar to those present in chromophobe renal cell carcinoma were present in rare tumor cells (Fig. 3D). Some tumor cells contained a significant amount of glycogen, but lipid droplets were not common. The tumor cells were ultra-structurally comparable to those of the original tumor, which had also been examined ultra-structurally, and which had shown areas reminiscent of oncocytoma and chromophobe cell carcinoma.

3.4 SKY analysis

Figure 4 displays the composite karyotype of UOK 257 (passage 18). The cell line is hypotriploid, with chromosome numbers ranging from 57–68 and a modal chromosome number of 65. The karyotype for UOK 257 is: 57~68<3n->,XX,−Y,+1,del(1)(p12)X2,del(3)(p12)X2,−4,+5,+6,der(6)t(6;17)(p11.2;q11.2)inv(6)(p25p11.2), der(6)t(6;17)(p11.2;q11.2)inv(6)(p25p11.2)ish t(6;8)(q27;q21)(MYC+), der(7)t(7;10)(q11.2;p12), ish+8(MYC+),−9,+10,der(10)t(7;10)(q11.2;p11.2),der(10)t(1;7)(?;q32)t(7;10)(q11.2;p11.2),+12,−13,−14,+16,der(11;16)(p10;p10)[2],−18,−19,+20,−22[cp15]. SKY analysis reveals whole chromosome gains for 1, 5, 6, 8, 10, 12, 16, and 20, and whole chromosome losses for Y, 4, 9, 13, 14, 18, 19, and 22. SKY detected partial gains of 1q, 8q21->8qter (MYC+), and 10q, and partial losses for 1p, and 3p. In addition, UOK 257 displayed an overall gain for 17q (four copies) and loss of 17p (two copies) due to the presence of two normal copies of chromosome 6 plus two unbalanced translocations; der(6)t(6;17) and der(6)t(6;8;17).

3.5 FISH with specific gene probes

Figure 5 depicts a metaphase spread analyzed by FISH, using a probe for the MYC oncogene on chromosome 8. The copy number for MYC in UOK 257 is heterogeneous, with some metaphase spreads containing three, four, and up to five copies, including the unbalanced translocation der(6)t(6;8;17).

3.6 Sequencing Analysis

We have confirmed the germline FLCN gene mutation from the primary UOK 257 cell line and in the single subcloned cell lines. The tumor lines showed the insertion of a “C” in nucleotide 1733 as shown in a sequencing chromatogram of DNA as previously described by us[7]. This is the same mutation that had been found in the patient’s germline. Notably, in the tumor lines, only the germline mutant copy is retained; the wild type copy is lost (data not shown). Furthermore, the patient’s germline mutation was present in the DNA from the implanted xenografts, confirming that the tumors were human in origin (data not shown). Since the UOK 257 cell line was established from a predominant clear cell type of tissue, we have examined VHL gene mutation status, and it demonstrated wild type VHL gene sequences retained in this cell line.