Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-ß1 heterozygous mice

Yang Kang1, Laurent L. Ozbun1, Jerry Angdisen1, Terry W. Moody1, Margaret Prentice1, Bhalchandra A. Diwan2 and Sonia B. Jakowlew1,3

1 National Cancer Institute, Cell and Cancer Biology Branch, Rockville, MD 20850
2 Intramural Support Program, SAIC-Frederick, Frederick, MD 21702, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We developed the AJBL6 transforming growth factor-beta 1 (TGF-ß1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-ß1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-ß1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15Ink4b and p27Kip1 decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16Ink4a/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-ß1 HT mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy.

Abbreviations: BrdU, bromodeoxyuridine; CDK, cyclin-dependent kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HT, heterozygous; LCM, laser capture microdissection; MMLV, Molony murine leukemia virus; NNK, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone; PCR, polymerase chain reaction; pRb, retinoblastoma protein; RT, reverse transcription; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulfate; TGF-ß RI, TGF-ß type I receptor; TGF-ß RII, TGF-ß type II receptor; TGF-ß, transforming growth factor-beta; WT, wildtype.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Little information is available concerning the early genetic events that occur in human neoplastic lung lesions. The location and late appearance of most lung cancers prevent comprehensive analyses of the early events associated with lung cancer. To circumvent the problems associated with obtaining early human lung cancer specimens, mouse model systems of lung cancer have been developed. Lung tumorigenesis in the A/J mouse model system has been extensively analyzed (1). The A/J mouse model system enables characterization of the molecular events in progressive stages of lung tumorigenesis with the development of hyperplasias, adenomas, and adenocarcinomas. Lung tumorigenesis in A/J mice can be induced by a variety of chemical carcinogens, including ethyl carbamate and its metabolite, vinyl carbamate (1,2). The transforming activity of DNA isolated from these tumors has been reported to be due in part to activated Ki-ras genes (3). However, because activation of Ki-ras alone cannot account totally for tumor susceptibility in A/J mice, additional genetic alterations are probably involved.

Multiple genes have been shown to be involved in activating tumorigenesis and in regulating the cell cycle. The cell cycle is regulated by a family of protein kinases composed of regulatory cyclin subunits and their catalytic cyclin-dependent kinase (CDK) partners. Different combinations of CDK and cyclin subunits operate at control checkpoints during the cell cycle to integrate mitogenic and antiproliferative signals that converge at these checkpoints. Cell proliferation and differentiation are specifically controlled in the G1 phase and the G1/S phase transition in the cell cycle (4,5). Several polypeptide growth factors exert mitogenic effects during the G1 phase by modulating CDK activities and by inducing specific cyclins, including transforming growth factor-beta 1 (TGF-ß1) (6). The ability of a cell to maintain control of its proliferative status is necessary for normal growth and homeostasis. Deregulation of genes that are involved in the progression from G1 to S phase of the cell cycle is a frequent event in several tumor types, including cancers of the lung, esophagus, and head and neck (7–9). The transition from G1 to S phase is regulated by the interaction of proteins that mediate the phosphorylation of retinoblastoma protein (pRb). The cyclin D1/CDK4/6 complex phosphorylates pRb, while p16Ink4a, a CDK inhibitor that through its interaction with CDK4, inhibits this process. Other proteins have also been shown to affect pRb, including the tumor suppressor genes p15Ink4b, p21Cip1, and p27Kip1. p15Ink4b specifically blocks the activation of CDK4 and CDK6 by inhibiting cyclin D/CDK complex formation, whereas p21Cip1 and p27Kip1 stabilize the assembly of cyclin D/CDK4 complexes. Phosphorylation of pRb results in the release of transcription factors, such as members of the E2F family, which are involved in the activation of genes that are necessary for entry into S phase. In addition to loss of pRb, unchecked phosphorylation of pRb due to loss of p16Ink4a or overexpression of cyclin D1 or CDK4 may lead to tumorigenesis (10–12). Amplification and overexpression of the cyclin D1 and CDK4 genes have been reported in a variety of human cancers, including lung cancer (13–16). Overexpression of cyclin D1 has also been shown to be a frequent event in bronchial preneoplasia and precedes the development of squamous cell carcinoma (17). Expression of cyclin D1 and CDK4 proteins has been shown to increase in lung tumors in A/J mice that have been challenged with carcinogen, while p16Ink4a expression was reduced in these tumors (18). In addition, overexpression of cyclin D1 has been shown to reduce TGF-ß type II receptor (TGF-ß RII) expression and growth inhibition by TGF-ß1 (19).

We have previously examined the role of the TGF-ß ligands and receptors in mouse lung tumorigenesis, and reported that while expression of the three TGF-ß ligands and the TGF-ß type I receptor (TGF-ß RI) was maintained in ethyl carbamate-induced lung tumors, that of TGF-ß RII was reduced in these tumors (20,21). More recently, we investigated the effect of reduced gene dosage of TGF-ß1 in diethylnitrosamine- and ethyl carbamate-induced mouse tumorigenesis. We reported enhanced lung and liver tumorigenesis in C57BL/6 mice heterozygous (HT) for the TGF-ß1 gene compared with wildtype (WT) littermates (22). In addition, we showed that lung tumors in AJBL6 TGF-ß1 HT mice, developed by mating A/J and C57BL/6 TGF-ß1 HT mice, had increased tumor incidence and multiplicity and decreased tumor latency compared with lung tumors in AJBL6 TGF-ß1 WT mice, with lung adenocarcinomas appearing by 4 mo after ethyl carbamate administration compared with 12 mo in AJBL6 TGF-ß1 WT mice. Use of the AJBL6 TGF-ß1 HT mouse makes it possible for us to obtain lung lesions at progressive stages of tumorigenesis, including hyperplasia, adenoma, and adenocarcinoma, readily and in quantity (23). Here, we have extended our study of carcinogen-induced mouse lung tumorigenesis in AJBL6 TGF-ß1 HT mice and examined cell cycle genes in ethyl carbamate-induced lung lesions. We report that altered expression of several G1/S regulatory genes is an early and frequent event in ethyl carbamate-induced lung lesions in AJBL6 TGF-ß1 HT mice, with expression of these genes progressively changing as lung carcinogenesis proceeds.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice and chemical carcinogenesis
Mouse lung tumors in AJBL6 mice that were induced by ethyl carbamate were available from a previous study (23). Briefly, C57BL/6 mice that were heterozygous for deletion of one TGF-ß1 allele (TGF-ß1+/-) were backcrossed one generation into pure A/J mice to obtain AJBL6 TGF-ß1 HT mice and their WT littermates. Genotyping was performed on DNA isolated from a section of the tails of F1 generation AJBL6 mice by using polymerase chain reaction (PCR) amplification with specific TGF-ß1 oligonucleotide primers that have been designed to distinguish TGF-ß1 WT and HT genotypes (24). Mice were housed in a pathogen-free barrier facility, maintained on a 12 h light–12 h dark cycle, and cared for according to National Institutes of Health guidelines. At 6–8 weeks of age, male and female mice of both genotypes were either given a single intraperitoneal injection of 1 mg/g ethyl carbamate (Sigma Chemical Co., St Louis, MO) in 0.1 ml of saline, or saline alone. Twenty mice were killed at monthly intervals after the injection for 12 mo. Lungs were removed, and one lung was immersed into either 10% neutral-buffered formalin or 70% ethanol at 4°C for 20 h, dehydrated, embedded in paraffin, and cut into 5 µm sections, and the other lung was frozen at –80°C. The sections were mounted onto slides either treated with poly-L-lysine for immunohistochemical staining or uncoated for laser capture microdissection analyses, respectively. For the study reported here, lungs from mice killed at 4, 6, 8, and 12 mo after ethyl carbamate administration were used.

Laser capture microdissection and RNA isolation
Laser capture microdissection (LCM) was used to cut out a small region from a specimen under microscope observation by laser beam (25). The specimen, fixed overnight at 4°C in ethanol, paraffin-embedded and sectioned onto an uncoated glass microscope slide, was set on a microscope stage and observed by a camera from the upper side. The UV-laser microbeam (Arcturus, Mountain View, CA) used consisted of high beam precision (wavelength 337 nm), and was coupled to an inverted PixCell microscope via the illumination path. After selecting tissues of interest based on hematoxylin and eosin and immunohistochemical staining analyses, specific areas from unstained serial sections were traced and photolysed by the laser beam whose energy was controlled and adjusted by the filter, and transferred to a thin polymer film on a microcentrifuge tube cap. After incubating the microdissected sample with 100 µl of guanidine isothiocyanate and ß-mercaptoethanol at room temperature for 5 min, RNA was isolated by two methods that produced RNA of equal quality. In the first method, RNA was extracted with phenol-chloroform-isoamyl alcohol, carrier glycogen was added with sodium acetate, and precipitated with ethanol at –20°C. The RNA pellet was dissolved in RNase-free water, and DNase I reaction buffer, Rnasin (Promega, Madison, WI), and RNase-free DNase I (Invitrogen, Carlsbad, CA) were added, and incubated at 37°C for 30 min to remove any genomic DNA contamination. Phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation of the RNA was repeated as before. The RNA pellet was resuspended in 11 µl of RNase-free water. In the second method, RNA was extracted from the microdissected sample using the Absolutely RNA microisolation kit with a silica-gel matrix according to the manufacturer's directions (Stratagene, San Diego, CA). RNase-free DNase I was added to remove residual genomic DNA as before prior to the washing steps while the RNA was still bound to the silica-gel matrix. RNA was eluted from the matrix in 30 µl of RNase-free water.

cRNA synthesis
Double-stranded cDNA was generated from the total RNA that was extracted from microdissected lung tumors and normal lung using the SuperScript Choice System (Invitrogen) according to the manufacturer's directions. The resulting cDNA was extracted with phenol-chloroform and precipitated with ethanol. Air-dried cDNA was dissolved in 15 µl of 10 mM Tris-HCl and EDTA buffer, pH 7.6. cRNA was synthesized from the cDNA using T7 RNA polymerase linear amplification according to the method outlined in ref. 26. cDNA synthesis was generated from the resulting cRNA, and a second round of cRNA synthesis was performed using T7 RNA polymerase linear amplification in order to generate a sufficient amount of RNA for use in hybridization.

Evaluation of representation of RNA populations isolated with LCM
Reverse transcription polymerase chain reaction (RT-PCR) amplification was performed on cRNA that was generated using two rounds of T7 amplification using Ready-to-Go beads (Amersham Biosciences, Piscataway, NJ) and the Primer Sets kit (Stratagene) for amplification of mouse genes with different levels of abundance, including mouse transcription factor EF1{alpha} (high abundance), mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (medium abundance), and mouse ornithine decarboxylase (low abundance) to evaluate the representation of RNA populations that were extracted from microdissected lung adenocarcinoma and normal lung samples. The RT-PCR reaction products were separated by gel electrophoresis, stained with ethidium bromide, and photographed.

Hybridization of radiolabeled probes to macroarrays
Double-stranded cDNA probes were generated from cRNA that was synthesized using two rounds of T7 RNA polymerase amplification as described previously and used to hybridize to macroarrays using the GEArray kit according to the manufacturer's directions (Superarray, Bethesda, MD). The filters were washed twice with 2X SSC and 1% sodium dodecyl sulfate (SDS) for 20 min at 68°C, twice with 0.1X SSC and 0.5% SDS for 20 min at 68°C, and then exposed to XAR-5 X-ray film.

Competitive RT-PCR
Quantitation of mRNA levels was performed using a modification of competitive RT-PCR amplification with endogenous target and homologous competitor mRNAs as described in ref. 27. To ensure that the efficiency of reverse transcription (RT) and PCR amplification of endogenous target and competitor mRNAs was equivalent, deletion constructs were designed so that the competitor RNA products were 20 bp shorter than the endogenous target RNA products and could compete with the target gene for primer annealing and amplification. Two pairs of oligonucleotide primers were designed. One pair of primers was used for RT-PCR amplification of the endogenous target and synthetic competitor mRNAs so that the target and competitor RT-PCR products could be distinguished by differences in size. Another pair of primers was used for construction of the synthetic competitor RNA. The primers were designed to span an intron to distinguish legitimate cDNA products from possible contaminant genomic DNA-derived cDNA products. Primer sets were as follows:

For competitor construction:
Cyclin D1

Sense:5'-GCGTAATACGACTCACTATAGGGAGAGGAGAGATGTGGACAT-CTGAGGGTAGTGGCACCCGCAAAGAGG-3' (nucleotides 1010–1028 underlined; nucleotides 1055–1074 bold)

Antisense: 5'-CTGAGGGCCTAAGCTGTGGC (nucleotides 1550–1531)

CDK4

Sense: 5'-GCGTAATACGACTCACTATAGGGAGAGGAGTTGTACGGCTGATGGATGATCGGGACATCAAGGTCACC-3' (nucleotides 212–229 underlined; nucleotides 251–270 bold)

Antisense: 5'-TACCAGAGCGTAACCACCACAG-3' (nucleotides 539–518)

p15Ink4b

Sense: 5'-GCGTAATACGACTCACTATAGGGAGAGGAGCAAGCACCTTTGCTGCCTCCAAACGTCAGTATCAGTCTCG-3' (nucleotides 41–60 underlined, nucleotides 84–103 bold)

Antisense 5'-GATACCTCGCAATGTCACGG-3'(nucleotides 377–358)

p16Ink4a

Sense:5'-GCGTAATACGACTCACTATAGGGAGAGGAGACGGTGCAGATTCGAACTGCCCGCCCGGTGCACGACGCAG-3' (nucleotides 251–270 underlined; nucleotides 291–310 bold)

Antisense: 5'-TCTGCTCTTGGGATTGGCCG-3'(nucleotides 580–561)

p21Cip1

Sense:5'-GCGTAATACGACTCACTATAGGGAGAGGAGGAGCCACAGGC-ACCATGTCCCACAGGAGCAAAGTGTGCCG-3' (nucleotides 79–98 underlined; nucleotides 126–145 bold)

Antisense: 5'-AGGGCAGAGGAAGTACTGGG-3'(nucleotides 388–369)

p27Kip1

Sense:5'-GCGTAATACGACTCACTATAGGGAGAGGAGAGCCTGGAGCG-GATGGACGCCTGCAGAAATCTCTTCGGCC-3' (nucleotides 251–270 underlined; nucleotides 301–320 bold)

Antisense: 5'-CGGTCCTCAGAGTTTGCCTC-3' (nucleotides 600–581)

pRb

Sense:5'-GCGTAATACGACTCACTATAGGGAGAGGAGAGTACCAAGGTTGATAATGCGTTATGCACTCTACAGGA-3' (nucleotides 501–520 underlined; nucleotides 551–570 bold)

Antisense: 5'-TGACCTCTTCTGGGTGTTCG-3' (nucleotides 850–831)

For endogenous target and competitor amplification:
Cyclin D1 (target: 260 bp product; competitor: 240 bp product)

Sense: 5'-AGATGTGGACATCTGAGGG-3' (nucleotides 1010–1028)

Antisense: 5'-AGGGGTGATGCAGATTCTATC-3' (nucleotides 1270–1250)

CDK4 (target: 249 bp product; competitor 229 bp product)

Sense: 5'-TTGTACGGCTGATGGATG-3' (nucleotides 212–229)

Antisense: 5'-CGGTCCCATTACTTGTCAC-3' (nucleotides 460–442)

p15Ink4b (target: 240 bp product; competitor: 217 bp product)

Sense: 5'-AAGCACCTTTGCTGCCTCC-3' (nucleotides 41–60)

Antisense: 5'-GCAGCACGACAAGCCTGTCC-3' (nucleotides 281–262)

p16Ink4a (target: 240 bp product; competitor: 220 bp product)

Sense: 5'-ACGGTGCAGATTCGAACTG C-3' (nucleotides 251–270)

Antisense: 5'-TACACAAAGACCACCCAGCG-3' (nucleotides 490–471)

p21Cip1 (target: 240 bp product; competitor: 214 bp product)

Sense: 5'-GAGCCACAGGCACCATGTCC-3' (nucleotides 79–98)

Antisense: 5'-AGACCTTGGGCAGCCCTAGC-3' (nucleotides 318–298)

p27Kip1 (target: 240 bp product; competitor: 270 bp product)

Sense: 5'-AGCCTGGAGCGGATGGACGC-3' (nucleotides 251–270)

Antisense: 5'-CACCTTGCAGGCGCTCTTGG-3' (nucleotides 520–501)

pRb (target: 230 bp product; competitor: 200 bp product)

Sense: 5'-AGTACCAAGGTTGATAATGC-3' (nucleotides 501–520

Antisense: 5'-CAACTACACACAACATTAGC-3' (nucleotides 750–731)

The sense primer for construction of the synthetic competitor included a core GC clamp sequence, a T7 polymerase promoter sequence (italics) at the 5' end to permit T7 polymerase binding and initiation of transcription, the target sense primer sequence (underlined), and a downstream specific target sense sequence (bold) at its 3' end. The antisense primer used for construction of the synthetic competitor initiated synthesis from the target cDNA ~50 nucleotides downstream of the target antisense binding site to equalize the efficiency of reverse transcription of competitor RNA. Competitor cDNA was synthesized from total mouse lung RNA (1 µg) using Molony murine leukemia virus reverse transcriptase (Invitrogen) and oligo d(T), and amplified by PCR using competitor sense and antisense primers. A portion of this PCR product (0.5 µg) was transcribed in vitro into competitor RNA using the RT-PCR Competitor Construction Kit according to the manufacturer's directions (Ambion, Austin, TX) in the presence of [32P]ATP (NEN/DuPont). Pilot RT-PCR experiments were initially performed to approximate the number of copies of synthetic competitor required to amplify equivalent amounts of competitor and target PCR products and the linear range of amplification where reaction components were still in excess and the PCR products were accumulating at a constant rate. Competitive RT-PCR was performed in a single tube using Ready-to-Go RT-PCR beads for both reverse transcription and subsequent PCR. The RT-PCR products were subjected to electrophoresis on 2% agarose gels containing ethidium bromide. The intensity of the synthetic competitor PCR product was used to quantitate the amount of endogenous target PCR product by interpolation. The dilution where the intensities of target and competitor PCR products were equivalent to each other represented the determination of the amount of target from the known amount of competitor. The authenticity of the products was confirmed by Southern blot hybridization with nested internal primers and DNA sequencing. When different populations of RNA were compared, RT-PCR amplification of the endogenous 18S rRNA in each population was first performed. Assuming that RT-PCR amplification of 18S rRNA would proceed at the same rate in each RNA sample, based on the amount of 18S rRNA RT-PCR product, the concentration of total RNA in each sample was made equivalent before competitive RT-PCR of target and competitor RNA was performed.

Immunohistochemical analysis
For the immunohistochemical localization of cell cycle proteins in paraffin sections of mouse lungs, the avidin-biotin complex technique was employed (Vector Laboratories, Burlingame, CA) according to the manufacturer's directions. Affinity-purified polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and included cyclin D1 (C-20) for cyclin D1, CDK4 (C-22) for CDK4, p15 (K18) for p15Ink4b, p16 (M-156) for p16Ink4a, p21 (C-19) for p21Cip1, p27 (C-19) for p27Kip1, and pRb (M-153) for pRb. The lung sections were stained with each antibody at the same time using identical conditions. Following immunostaining, the tissues were viewed, analyzed, and photographed on a Nikon LaboPhot-2 microscope (Image Systems, Columbia, MD). A control for specificity included using primary antisera preincubated with a 20-fold excess of the appropriate peptide.

In vivo proliferation
Mice were injected intraperitoneally with 150 mg bromodeoxyuridine (BrdU) (Roche, Palo Alto, CA) per kg 1 h before they were killed. Ethanol-fixed lung sections were immunostained using an anti-BrdU monoclonal antibody (Dako, Carpintera, CA). After counterstaining with hematoxylin, the number of labeled cells in 1000 cells was determined for each lung sample.

Statistics
Statistical significance was determined using the Student's t-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Laser capture microdissection, RNA isolation, evaluation of representation of RNA populations, and macroarray analysis
To examine the mRNAs for cell cycle genes in lung tumors and normal lung tissue from AJBL6 TGF-ß1 HT mice, LCM under direct microscopic visualization was used to dissect lung adenocarcinoma and normal lung tissue separately from serial ethanol-fixed and paraffin-embedded sections of mouse lung specimens. Figure 1Go shows a representative illustration of lung adenocarcinomas and normal lungs before (Figure 1A and BGo) and after microdissection (Figure 1C, D, G, and HGo) and their capture on microcentrifuge tube caps (Figure 1E, F, I, and JGo). Adenocarcinomas were characterized as irregular, poorly circumscribed neoplasms that showed architectural distortion and variation in appearance and organization with increased mitotic activity. Figure 1EGo demonstrates the separation of adenocarcinoma from surrounding lung alveolar tissue by LCM. Figure 1FGo shows the separation of bronchiolar tissue from surrounding alveoli and adenocarcinoma. In addition, Figure 1HGo shows LCM of normal alveolar and bronchiolar tissue. Total RNA was extracted from 12 individual lung adenocarcinomas and normal lungs that were isolated using LCM from 12 AJBL6 TGF-ß1 HT mice that had been treated with ethyl carbamate or saline, and 50% of each RNA sample was pooled. Following conversion of the pooled RNAs that were extracted from microdissected samples to double-stranded cDNAs, sufficient amounts of cRNAs were generated using two rounds of T7 RNA amplification. In order to evaluate the representation of RNA populations in the cRNAs, RT-PCR amplification was performed on the cRNAs using primers for amplification of mouse genes with different known levels of abundance. Figure 2Go shows RT-PCR amplification of cRNA generated from whole normal lung tissue, microdissected normal lung containing alveolar and bronchiolar epithelial cells, and microdissected lung adenocarcinoma using primers for EF1{alpha}, GAPDH, and ornithine decarboxylase. The RT-PCR products for EF1{alpha}, GAPDH, and ornithine decarboxylase were observed in cRNA from whole normal lung, and microdissected normal lung and lung adenocarcinoma in similar amounts.



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Fig. 1. Laser capture microdissection of paraffin-embedded mouse lung tumor and normal lung tissues from AJBL6 TGF-ß1 HT mice. Lung adenocarcinomas and normal lung tissues (A, B) before microdissection; (C, D, G, H) after microdissection; and (E, F, I, J ) captured and adherent to the microcentrifuge tube cap. The illustrations shown here are representative of multiple microdissections.

 


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Fig. 2. RT-PCR amplification of cRNAs from whole normal lung, microdissected normal lung, and microdissected lung adenocarcinoma using primers for (lane 1) mouse EF1{alpha}, (lane 2) mouse GAPDH, and (lane 3) mouse ornithine decarboxylase. M, DNA molecular weight marker.

 
cDNA probes generated from cRNAs from microdissected lung tissues were used to hybridize to mouse cell cycle cDNA macroarrays. Figure 3Go shows representative macroarrays that were hybridized with cDNA probes generated from RNAs isolated from lung adenocarcinomas and normal lung tissue, and Table IGo shows that nine differentially expressed genes were identified from these macroarrays, including CDK4, cyclin D1, E2F1, p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb. While the expression of the genes for CDK4, cyclin D1, and E2F1 increased in lung adenocarcinomas, expression of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb decreased in comparison in these tumors. Interestingly, three cell cycle genes were not changed between lung adenocarcinomas and normal lung, including CDK6, CDC2, and cyclin B. In addition, a number of cell cycle genes were below the level of detection, including CDK2, cyclin A, cyclin C, cyclin D2, cyclin D3, cyclin E2, p18, p19, and Skp1p-Cdc53p-F-box protein complex proteins Skp1 and Skp2. The positive control genes that included ß-actin and GAPDH remained unchanged in tumor and normal lung, while pUC18 bacterial plasmid negative control DNA was not detected.



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Fig. 3. Cell cycle cDNA macroarray analysis. Mouse cell cycle cDNAs that were arrayed onto filter membranes were hybridized with radioactively labeled double-stranded cDNA probes synthesized from cRNAs generated from (A) lung adenocarcinomas and (B) normal lung tissue from 12 AJBL6 TGF-ß1 HT mice that were treated with either ethyl carbamate or saline. The spots that are boxed with dashed lines represent genes that are increased in lung adenocarcinomas compared with normal lung. The spots that are boxed with solid lines represent genes that are decreased in lung adenocarcinomas compared with normal lung. The filters shown are representative of three separate experiments.

 

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Table I. Cell cycle cDNA filter array expression
 
Competitive RT-PCR
Because the amounts of RNAs that we obtained from microdissected lung adenocarcinomas and normal lung tissue were limited, we could not use northern blot hybridization to verify differential expression of cell cycle genes obtained from macroarray analysis. Instead, competitive RT-PCR was used to verify, and also to quantitate, the mRNAs for the cell cycle genes that were demonstrated to be differentially expressed in lung adenocarcinomas and normal lung tissue from AJBL6 TGF-ß1 HT mice by macroarray analysis. Total RNA was extracted from 12 microdissected lung adenocarcinomas and normal lung tissue from 12 different AJBL6 TGF-ß1 HT mice. After RT-PCR was performed on 18S rRNA from each of the RNA samples, and the amounts of total RNA were equalized relative to 18S rRNA, competitive RT-PCR amplification was used to quantitate the amount of cell cycle mRNAs in lung adenocarcinomas and normal lung. Figure 4Go shows a representative ethidium bromide staining pattern of the endogenous and competitor cyclin D1 RT-PCR products from adenocarcinoma and normal lung tissue. Because the absolute amount of starting total RNA from tissues isolated by LCM cannot be accurately quantitated due to the limited quantities, only relative quantitations are presented. While the adenocarcinoma had at least 12.5 x 105 copies of cyclin D1 mRNA per aliquot of total RNA, the normal lung tissue had 6.25 x 105 copies in the same quantity of total RNA. In addition to cyclin D1, competitive RT-PCR was performed on the mRNAs for CDK4, p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb in lung adenocarcinomas and normal lung from 12 AJBL6 TGF-ß1 HT mice and values were averaged. Table IIGo shows ~2- and 3-fold increases in the number of copies of cyclin D1 and CDK4 mRNAs, respectively, and a contrasting 2- to 4-fold decrease in the number of copies of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb mRNAs in lung adenocarcinomas compared with normal lung tissue in these mice.



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Fig. 4. Quantitation of cyclin D1 mRNA in lung adenocarcinomas and normal lung tissue by competitive RT-PCR in AJBL6 TGF-ß1 HT mice. Total RNA was extracted from 12 individual lung adenocarcinomas and normal lung tissues isolated by laser capture microdissection from paraffin-embedded sections of lungs from AJBL6 TGF-ß1 HT mice. The concentrations of the total RNA samples were adjusted so that they were equal based on the level of 18S cDNA in each sample. Serial 1:2 dilutions of synthetic cyclin D1 competitor RNA starting from 50 x 105 copies to no mRNA copies underwent 30 cycles of PCR amplification. Following size fractionation by gel electrophoresis, the gel was stained with ethidium bromide and photographed. The dilution where the intensity of endogenous target (ET) is equal to that of the synthetic competitor (CT) indicates the amount of target mRNA. The ethidium bromide staining pattern is representative of the 12 lung samples.

 

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Table II. Quantitation of cell cycle mRNAs in lung adenocarcinomas and normal lung tissue in AJBL6 TGF-ß1 HT mice by competitive RT-PCR analysis
 
To determine the level of mRNAs of cell cycle proteins at different stages of lung tumorigenesis, LCM was used to isolate 12 samples of lung hyperplasias, adenomas and adenocarcinomas from 12 AJBL6 TGF-ß1 HT mice treated with ethyl carbamate and killed after 8 mo. Hyperplasias were characterized by increased cell number resulting from cell division with cellular and nuclear pleomorphism, while adenomas were characterized as small neoplasms representing cell proliferation that caused compression of the surrounding parenchyma and were well circumscribed. Total RNA was isolated from the microdissected hyperplasias, adenomas, and adenocarcinomas, and from normal lung tissue, and RT-PCR amplification of 18S rRNA was performed. After adjusting the concentrations of the different samples of RNA so that they were equivalent based on RT-PCR amplification of 18S rRNA, competitive RT-PCR amplification analysis was performed on cell cycle mRNAs in each sample, the means were determined, and then compared. Figure 5Go shows that the level of cyclin D1 and CDK4 mRNAs increased with progressive lung tumorigenesis, with hyperplasias having 50% more CDK4 mRNA than normal lung, adenomas having 22% more cyclin D1 and CDK4 mRNAs than hyperplasias, and adenocarcinomas having 27% and 65% more cyclin D1 and CDK4 mRNAs, respectively, than adenomas. In contrast, except for p27Cip1 mRNA which showed comparable levels in hyperplasias and normal lung, the level of the mRNAs for p15Ink4b, p27Cip1, and pRb decreased with progressive lung tumorigenesis, with hyperplasias, adenomas, and adenocarcinomas having 21–35%, 41–55%, and 63–76%, respectively, lower levels of these mRNAs than normal lung. Although pRb mRNA levels decreased in adenomas and adenocarcinomas compared with normal lung and hyperplasias, no significant difference was detected in the pRb mRNA transcript levels between adenomas and adenocarcinomas.



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Fig. 5. Quantitation of cell cycle mRNAs in normal lung and progressive lung tumorigenesis in AJBL6 TGF-ß1 HT mice by competitive RT-PCR. Total RNA was extracted from 12 individual normal lungs, lung hyperplasias, adenomas, and adenocarcinomas isolated by laser capture microdissection from ethanol-fixed and paraffin-embedded sections of lungs from AJBL6 TGF-ß1 HT mice. The concentrations of the total RNA samples were adjusted so that they were equal based on the level of 18S cDNA in each sample. Serial 1:2 dilutions of synthetic competitor RNA for cyclin D1, CDK4, p15Ink4b, p27Cip1, and pRb underwent 30 cycles of PCR amplification. Following size fractionation by gel electrophoresis, the gels were stained with ethidium bromide and photographed. The mean of the dilutions where the intensity of endogenous target is equal to that of the synthetic competitor indicating the amount of target mRNA ± standard error is presented. (open bar) normal lung (N); (gray bar) hyperplasia (H); (hatched bar) adenoma (A); (solid bar) adenocarcinoma (C). *P < 0.05; **P < 0.01.

 
Immunohistochemical analysis
To examine the distribution of cell cycle proteins in malignant and normal mouse lung tissue from AJBL6 TGF-ß1 HT mice treated with ethyl carbamate or saline, indirect immunoperoxidase staining studies were performed. Figure 6Go illustrates the immunohistochemical staining pattern of cell cycle proteins in sections of lung adenocarcinoma and normal lung from an AJBL6 TGF-ß1 HT mouse killed 8 mo after ethyl carbamate administration. Moderate to strong nuclear staining for cyclin D1 and CDK4 was detected in the adenocarcinoma (Figure 6A and BGo) and in 11 additional lung adenocarcinomas from AJBL6 TGF-ß1 HT mice, with >80% of the tumor cells showing expression of cyclin D1 and CDK4 (Table IIIGo). All of the AJBL6 TGF-ß1 HT mice that were examined had lung adenocarcinomas. There were occasional small focal areas that showed weaker staining for cyclin D1 or CDK4 than the majority of the tumor in some of the adenocarcinomas. Strong immunostaining for cyclin D1 and CDK4 was also observed in adenocarcinomas obtained from AJBL6 TGF-ß1 HT mice treated with ethyl carbamate for either 4, 6, or 12 mo, and also in adenocarcinomas obtained from AJBL6 TGF-ß1 WT mice that were killed 12 mo after ethyl carbamate administration (data not shown). Interestingly, similar nuclear staining distribution patterns for cyclin D1 and CDK4 were also detected in lung adenocarcinomas of AJBL6 TGF-ß1 WT mice (Table IIIGo). In contrast, Figure 6A and BGo insets show that only weak staining for cyclin D1 and CDK4 was detected in fewer than 20% of cells of normal lung tissue from these mice (Table IIIGo). Both normal lung bronchiolar and alveolar epithelial cells showed similar weak immunostaining patterns for cyclin D1 and CDK4. Immunohistochemical staining for cyclin D1 and CDK4 was also performed on lung adenomas and hyperplasias from AJBL6 TGF-ß1 HT and WT mice. The amount of nuclear staining for cyclin D1 and CDK4 increased in adenocarcinomas compared with adenomas and hyperplasias of mice of either TGF-ß1 genotype. Immunostaining for both cyclin D1 and CDK4 increased progressively from normal lung on to adenocarcinomas, with hyperplasias showing a greater percentage of cells staining positive compared with nomal lung, adenomas showing a greater percentage of cells staining positive compared with hyperplasias, and adenocarcinomas showing even a greater percentage of cells staining than adenomas in a majority of AJBL6 TGF-ß1 HT mice (Table IIIGo). Immunohistochemical staining for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb was also performed on normal lung and lung adenocarcinomas of AJBL6 TGF-ß1 HT and WT mice. Figure 6C–HGo and Table IIIGo show strong immunostaining for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb in lung bronchiolar tissue of AJBL6 TGF-ß1 HT mice, but only weak staining for these proteins in alveolar tissue, with >60% of cells staining positive, and a decrease in staining to <40% of cells staining positively in lung adenocarcinomas in a majority of mice; the level of staining for these proteins was similar in lung lesions of AJBL6 TGF-ß1 WT mice, except for pRb, which showed more cells staining positively for pRb in adenocarcinomas of AJBL6 TGF-ß1 WT mice than in HT mice. Both nuclear and cytoplasmic staining for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb was observed in adenocarcinomas. Adenocarcinomas also showed occasional small focal areas of cells that stained negatively for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, or pRb, and other areas of single and multiple cells that stained strongly positive for some of these cell cycle proteins within the focal areas showing only weak staining. None of the positively staining single or multiple cells within the weakly staining areas of the adenocarcinomas were positive for all of the five tumor suppressor gene proteins that were examined. Immunostaining for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb was observed in <60% of the cells in lung adenomas of both AJBL6 TGF-ß1 HT and WT mice, except for pRb. Similar to adenocarcinomas, more cells in adenomas of AJBL6 TGF-ß1 WT mice showed staining for pRb than in AJBL6 TGF-ß1 HT mice (Table IIIGo). Interestingly, fewer cells stained positively for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb in lung hyperplasias of AJBL6 TGF-ß1 HT mice than in normal lung tissue, while comparable staining of p27Kip1 and pRb occurred in normal lung and hyperplasias of AJBL6 TGF-ß1 WT mice (Table IIIGo). Like adenocarcinomas, some adenomas had focal areas that showed negative staining for at least one of the p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb proteins, although these areas were considerably smaller and occurred even less frequently than in adenocarcinomas (data not shown). To demonstrate specificity of the antibody, Figure 6DGo inset shows lack of staining for p16Ink4a in normal lung tissue when the p16Ink4a antibody was preincubated with the antigenic peptide before immunostaining was performed. Similar absence of immunostaining in normal lung tissue was observed for p15Ink4b, p21Cip1, p27Kip1, and pRb when the antibodies were preincubated with their respective antigenic peptides before immunohistochemical staining was performed (data not shown). Figure 6HGo shows positive immunostaining for p16 Ink4a in normal lung bronchiolar tissue and only weak staining in alveolar tissue. Similar positive immunostaining for p15Ink4b, p21Cip1, p27Kip1, and pRb proteins was also observed in normal lung bronchiolar tissue (data not shown). In addition, proliferation was determined in lung adenocarcinomas and normal lung by BrdU incorporation. Figure 6Go shows intense nuclear immunostaining for BrdU in lung adenocarcinomas, while only weak staining was detected in normal lung (Figure 6Go inset). While an average of 42% of cells were labeled with BrdU in lung adenocarcinomas, with some adenocarcinomas having as little as 13% or as many as 75% of cells staining positive for BrdU, <1% of cells labeled with BrdU in normal lung tissue.



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Fig. 6. Immunohistochemical staining analysis of cell cycle proteins in lung adenocarcinomas and normal lung tissues from AJBL6 TGF-ß1 HT mice. Mouse lung specimens from AJBL6 TGF-ß1 HT mice 8 mo after administration of (A–G, I) ethyl carbamate or (H, insets in A, B, D, I) saline were collected, fixed in formalin, embedded in paraffin, and sectioned onto microscope slides, were reacted with antibodies against (A) cyclin D1, (B) CDK4, (C) p15Ink4b, (D) p16Ink4a, (E) p21Cip1, (F) p27Kip1, (G) pRb, (H) p16Ink4a, and (I) BrdU. In (D inset), the antibodies against p16Ink4a were preincubated with an excess of immunogen before immunostaining. The immunostaining is representative of 12 separate lung samples. Magnification: (A, B) 400x; (B inset) 200x; (C–G, A inset, D inset) 100x; (I) 40x; (H, I inset) 20x.

 

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Table III. Immunohistochemical staining distribution scoring for normal lung and lung lesions in AJBL6 TGF-ß1 HT and WT mice
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Deregulated signaling pathways governing the cell cycle have been recognized as an important factor in tumorigenesis. An increasing number of alterations in the normal expression patterns of proteins in these pathways, including cyclin D1, p16Ink4a, and pRb, have been reported in multiple human tumors (28). In the present study, we examined expression of the cyclin D1/CDK4/p16Ink4a/pRb signaling pathway components in the AJBL6 TGF-ß1 HT mouse model that we recently generated to investigate carcinogen-induced lung tumorigenesis (23). We demonstrate that by combining and integrating LCM, cRNA, and cDNA macroarrays, one can successfully screen various lung lesions and normal lung obtained in situ from fixed tissue and subsequently identify differential gene expression. Laser capture microdissection was used to isolate lung adenocarcinomas and normal lung tissue, thereby minimizing, if not eliminating, heterogeneous cellular elements, and RNA was extracted from the microdissected tissues. Following amplification of the microdissection-derived RNA to cRNA and hybridization to macroarrays containing mouse cell cycle cDNAs, it was determined that nine cell cycle genes are differentially regulated in ethyl carbamate-induced lung adenocarcinomas and normal lung tissue in AJBL6 TGF-ß1 HT mice, including cyclin D1, CDK4, E2F1, p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb. According to macroarray analysis, while expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas of AJBL6 TGF-ß1 HT mice, expression of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb decreased in comparison. Increased expression of cyclin D1 and CDK4 and decreased expression of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb in mouse lung tumor cells would likely provide these cells with a selective growth advantage. Several studies have suggested oncogenic roles for cyclin D1 and CDK4 and tumor suppressor roles for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, and pRb. Amplification and overexpression of the cyclin D1 and CDK4 genes has been reported in a variety of human cancers, including lung cancer, and are frequent events in bronchial preneoplasia that precedes the development of squamous cell carcinoma (13–17,29). Immunohistochemical staining and western blot analyses showed overexpression of cyclin D1 and CDK4 proteins in 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung adenocarcinomas and increased expression of CDK4 in N-methyl-N-nitrosourea-induced rat mammary tumors and in intestinal adenomas from Min mice (18,29,30). Our study showing increased expression of cyclin D1 and CDK4 in lung carcinogenesis using the AJBL6 TGF-ß1 HT mouse model is in agreement with earlier mouse lung carcinogenesis studies, and extends these studies by showing altered expression of these genes and their proteins in progressive lung carcinogenesis, with increased expression of these genes beginning to occur early in hyperplasias and adenomas. Studies of in vivo and in vitro effects of cyclin D1 have shown its involvement in proliferation. Overexpression of cyclin D1 in rodent fibroblasts and transgenic mice enhances their tumorigenicity and proliferation in nude mice and results in hyperplasia and in tumors of the mammary epithelium (31,32). In addition, transfection of antisense cyclin D1 cDNA into mouse lung tumor cells, and human esophageal and colon cancer cell lines has been shown to reduce their proliferation rate in vitro and their tumorigenic potential in nude mice (33–35). Immunostaining for BrdU showed intense nuclear staining in adenocarcinomas compared with weak staining in normal lung in our study, indicating increased cell proliferation in adenocarcinomas that is consistent with earlier studies (36,37). Increased immunostaining for BrdU in adenocarcinomas is also consistent with increased cyclin D1 and CDK4 expression in adenocarcinomas.

In contrast to increased cyclin D1 and CDK4 levels in lung carcinogenesis, homozygous co-deletion and decreased expression of p15Ink4band p16Ink4a has been shown to occur in some mouse lung tumor cells in vitro (38). Reduced or absent staining of p16Ink4a, and of pRb staining as well, has also been reported in NNK- and methylene chloride-induced mouse adenocarcinomas, which interestingly, also showed areas of multiple and single cells positively stained for p16Ink4a in the areas of negative staining, similarly to the study that we are reporting here (18,39). Inactivation of p16Ink4a has been reported to occur in the progression of mouse lung tumors to malignancy (18,40–42), and in human lung, esophageal, and head and neck cancers as well (7–9). In our study, the percentage of cells that showed positive staining for p16Ink4a in adenocarcinomas was significantly lower than in normal lung, and a few focal areas in adenocarcinomas showed almost complete absence of staining for p16Ink4a. A model of lung tumorigenesis has been reported in which it has been proposed that overexpression of cyclin D1 may be an early event, followed either by loss of p16Ink4a or pRb expression, which reflects a functional redundancy of p16Ink4a and pRb on a common p16Ink4a/pRb regulatory pathway (42). Our study of lung carcinogenesis in AJBL6 TGF-ß1 HT mice agrees with this proposed model of lung tumorigenesis, with reduced expression of p16Ink4a and pRb in progressive lung carcinogenesis. In addition, we showed that loss of pRb expression is an earlier event in AJBL6 TGF-ß1 HT mice than in AJBL6 TGF-ß1 WT mice, with lung hyperplasias having decreased pRb levels compared with normal lung that decreased to even lower levels in adenomas and adenocarcinomas in AJBL6 TGF-ß1 HT mice, whereas lung hyperplasias and adenomas in AJBL6 TGF-ß1 WT mice showed pRb levels that were comparable with normal lung. The earlier loss of pRb that occurs in lung hyperplasias and adenomas of AJBL6 TGF-ß1 HT mice compared with AJBL6 TGF-ß1 WT mice is the most striking difference that occurs among the cell cycle genes when AJBL6 TGF-ß1 HT and WT mice are compared. It is possible that reduced TGF-ß1 gene dosage in AJBL6 TGF-ß1 HT mice may result in localized reduced p16Ink4a expression below threshold levels in tissue areas, which may then lead to unchecked phosphorylation of pRb, and ultimately, to increased lung tumorigenesis. Future studies will need to address these possibilities at the molecular level.

In addition to p16Ink4a and pRb, we also examined p21Cip1 and p27Kip1 tumor suppressors in mouse lung carcinogenesis that also showed differential expression in lung adenocarcinomas compared with normal lung in AJBL6 TGF-ß1 HT mice by macroarray analysis. The occurrence of decreasing levels of p21Cip1 and p27Kip1, as well as of p15Ink4b, p16Ink4a, p57Kip2, and pRb expression at the same time and in many of the same lung tumors suggests possible cooperation of these genes during lung tumorigenesis in vivo. Such cooperation might parallel the findings of earlier in vitro studies where, for example, p21Cip1 and p27Kip1 together, and p15Ink4band p27Kip1 together, were shown to cooperate to induce cell cycle arrest in response to TGF-ß1, and their coordinate subcellular locations contribute to inhibition of CDK4 and CDK2 (11,43,44), and the demonstration of multiple components of the pRb pathway being simultaneously differentially affected in non-small cell lung tumors in vivo (45). Interestingly, while the immunohistochemical staining distribution for cyclin D1 and CDK4 increased, and those for p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb decreased as lung tumorigenesis progressed from hyperplasias to adenocarcinomas in AJBL6 TGF-ß1 HT mice, this was not always reflected by similar changes in the corresponding mRNA levels as determined by competitive RT-PCR. For example, although expression of cyclin D1 protein increased in hyperplasias, adenomas, and adenocarcinomas in AJBL6 TGF-ß1 HT mice compared with normal lung, the level of cyclin D1 mRNA did not change significantly in hyperplasias and adenomas compared with normal lung. Similarly, while pRb protein decreased in adenocarcinomas compared with adenomas, this was not reflected by the pRb mRNA transcript levels. Discrepancies between mRNA and protein levels have been reported in human and mouse tumors (39,46,47) and have been attributed to such processes as differential turnover rates for mRNA and protein, short-lived mRNA transcripts, post-translational modification, proteosomal degradation, and tissue heterogeneity.

Identifying genes that are differentially expressed in early human lung lesions compared with their normal counterpart is a challenging issue because of the difficulty in diagnosing and obtaining human lung lesions when they are in the early stages of the disease. Use of the AJBL6 TGF-ß1 HT mouse model system has allowed us not only to examine cell cycle proteins in early lung tumorigenesis, but also to measure the levels of the mRNAs of these cell cycle proteins during progressive lung carcinogenesis. The mRNA levels of components of the cyclin D1/CDK4/p16Ink4a/pRb signaling pathway were measured by competitive RT-PCR analysis in normal mouse lung and in progressively developing lung lesions isolated by laser capture microdissection. We observed significant increases in cyclin D1 and CDK4 mRNA levels and decreases in p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb mRNA levels in lung adenocarcinomas when compared with normal lung tissue, thus validating our macroarray findings. In addition, we measured progressive increases in cyclin D1 and CDK4 mRNA levels and contrasting progressive decreases in p15Ink4b, p27Kip1, and pRb mRNA levels as lung tumorigenesis develops from hyperplasias on to adenocarcinomas. Immunohistochemical analysis showed increasing numbers of tumor cells that showed positive staining for cyclin D1 and CDK4 proteins in progressive tumorigenesis with increasing mRNA levels of these corresponding proteins and increased cell proliferation in lung tumor cells compared with normal lung cells in AJBL6 TGF-ß1 HT mice. This suggests that increased expression of cyclin D1 and CDK4 may be linked to increased proliferation in lung tumor cells. Our observation of increased expression of the mRNAs and proteins for cyclin D1 and CDK4 in lung adenocarcinomas with increasing proliferation in AJBL6 TGF-ß1 HT mice parallels other mouse tumorigenesis models. Overexpression of cyclin D1 and CDK4 has been shown to be associated with increased cell proliferation (35,48), and transgenic mice with cyclin D1 overexpression show increased cell proliferation in targeted tissues (48). Since cyclin D1 and CDK4 are among the genes that are essential for controlling the progression of the G1 phase to the S phase and the onset of DNA replication, increased expression of cyclin D1 and CDK4 may cause or contribute to abnormalities in the control of cell growth and proliferation rate. In vivo, cyclin D1 overexpression has been found in invasive carcinomas, including lung cancer and head and neck squamous cell carcinoma (15–17). Levels of cyclin D1 expression were shown to be 4- to 100-fold higher in many non-small cell lung carcinomas than in normal bronchioepithelial cells (49). Although the cyclin D1 gene was shown not to be amplified or rearranged in a cohort of non-small cell lung cancer patients, cyclin D1 expression was found to correlate with altered p53 protein expression, but not with p16Ink4a or Rb protein status, and patients with cyclin D1 positive tumors survived longer than patients with tumors that were negative for cyclin D1 (49). Overexpression of cyclin D1 has also been reported to frequently occur in bronchial preneoplasia (17), indicating that this is a relatively early event in tumorigenesis. Manipulation of cyclin D expression has shown why overexpression of this gene confers a growth advantage in tumor cells. Ectopically overexpressed cyclin D1 can accelerate the rate of the cell cycle of primary rat fibroblasts, and in the same cells, can cooperate with activated ras to induce transformation (50). Administration of antisense cyclin D1 RNA in a variety of tumor cell types results in the retardation of cell growth in culture and loss of tumorigenicity in vivo (33–35,51). Our study showed increased expression of cyclin D1 and CDK4 in carcinogen-induced mouse lung tumors compared with normal lung tissue, and suggests that the cyclin D1 and CDK4 genes, and the proteins that they encode, may play an important role in mouse lung carcinogenesis induced by ethyl carbamate. Interestingly, deletion of one TGF-ß1 allele does not appear to affect expression of the mRNAs and proteins for these cell cycle genes differentially in mouse lung tumorigenesis compared with wildtype mice except for the earlier loss of pRb in hyperplasias and adenomas of AJBL6 TGF-ß1 HT mice compared with their wildtype littermates. This finding was unexpected since one of the most striking in vitro effects of TGF-ß1 is the reversible growth arrest of non-neoplastic epithelial cells in the G1 phase of the cell cycle. Several mechanisms involving CDKs and CDK inhibitors have been described to explain the inhibitory activities of TGF-ß1 in vitro (52). TGF-ß1 suppresses CDK4 synthesis in mink lung epithelial cells and keratinocytes (53,54). TGF-ß1 increases the levels of the CDK inhibitor p15Ink4b in mink lung epithelial cells (53), and additional CDK inhibitors, including p21Cip1 and p27Kip1 in keratinocytes and pancreatic cancer cells (55–58). TGF-ß1-induced inhibition of epithelial cell proliferation is associated with a reduction of cyclin D1 expression in different cell types in culture (59–62). However, the ability of TGF-ß1 to affect growth depends on multiple conditions, including cell type, cell density, growth conditions, and the presence or absence of other growth factors, so that these effects may not be the same when examined under in vivo circumstances as under in vitro conditions where influences from the environment, including extracellular matrix proteins, proteases, and integrins to name just a very few proteins, are important factors in influencing the ability of TGF-ß1 to act (63,64). In addition, although TGF-ß1 inhibits the proliferation of most non-neoplastic cells in culture, it has been demonstrated that many neoplastic cells have become resistant to the growth inhibitory effects of TGF-ß1 (63,64). It may be possible that reduced gene dosage of TGF-ß1 has no effect on cell cycle genes in lung adenocarcinomas that may have acquired resistance to TGF-ß1 or in premalignant cells in hyperplasias and adenomas that may be becoming resistant to the growth inhibitory effects of TGF-ß1. Enhanced susceptibility to lung tumorigenesis induced by ethyl carbamate in AJBL6 TGF-ß1 HT mice does not appear to involve selective stimulation of tumors with differential patterns of expression of cyclin D1 and CDK4 or of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and pRb compared with AJBL6 TGF-ß1 WT mice. Apparently there are other genes and/or factors that contribute to enhanced lung tumorigenesis in the AJBL6 TGF-ß1 HT mouse that will need to be identified.


    Notes
 
3 To whom correspondence should be addressed at Email jakowles{at}mail.nih.gov Back


    Acknowledgments
 
The authors thank Dr L.Wakefield (NCI, Laboratory of Cell Regulation and Carcinogenesis) for supplying C57BL/6 TGF-ß1 HT and WT mice. They also acknowledge the assistance of Ke Xiao (Montgomery Blair High School, Silver Spring, MD).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 19, 2001; revised March 8, 2002; accepted March 22, 2002.