Transient alterations in the expression of protease and extracellular matrix genes during metastatic lung colonization by H-ras-transformed 10T1/2 fibroblasts

It has been proposed that tumor progression is a selective process and that only a minority of tumor cells survive this selection because they possess the phenotypic traits necessary for metastasis and organ colonization. Both proteases and extracellular matrix proteins have been implicated in invasion and metastasis formation. To examine the nature of the selection process, we transformed 10T1/2 fibroblasts with T24 H-ras and the neoR gene and selected a clonal line expressing the mutant ras gene. After i.v. injection of this line into syngeneic C3H/HeN mice, tumor cells were recovered from lungs by enzymatic treatment and selective outgrowth in G418. Less than one of 10(3) cells survived in the lung 30 min after inoculation, and these exhibited a unique phenotype. This was characterized by a propensity to lodge in the lung on reinjection; markedly enhanced mRNA levels of procollagen alpha 2(I), procollagen alpha 1(III), and fibronectin; and decreased levels of laminin, major excreted protein (procathepsin L), transin, and H-ras. Between 1 and 9 days after tumor injection, the phenotype of the cells surviving in the lung changed dramatically and exhibited a pattern of gene expression with increased protease and low matrix protein mRNA levels. This coincided with a 26-fold increase in the ability to colonize lungs on i.v. injection. Both the phenotype characterized by its propensity to arrest in the lung and that showing enhanced metastatic ability were unstable on prolonged in vitro culture. We hypothesize that two selection events have occurred. The first is for lung arrest and implantation of variants of the injected tumor with high matrix protein and low protease levels. A second selection then occurs for tumor cells that carry a favorable phenotype for invasion and proliferation which is associated with low matrix protein and high protease gene expression. These two phenotypes are represented within a clonal population of recently transformed tumor cells.     

The inhibition of platelet aggregation of metastatic H-ras-transformed 10T1/2 fibroblasts with castanospermine, an N-linked glycoprotein processing inhibitor.

A series of T24-H-ras-transformed 10T1/2 fibroblasts with varying metastatic potential was tested for the ability to aggregate platelets. Results indicate that although platelet activation was always detected in the highly metastatic cells, some non-metastatic cells also have the ability to cause platelet aggregation, suggesting that this is a necessary but not sufficient characteristic of the metastatic phenotype. Apyrase, an ADP scavenger, effectively inhibited platelet aggregation by metastatic cells, however, there was no significant increase in ADP secretion or relation to the ability of the tumor cells to activate platelets. Hirudin, a thrombin inhibitor, did not affect aggregation, suggesting that the pathway of activation is thrombin-independent. The glycoprotein processing inhibitor, castanospermine, which reduces glycosidase I activity and metastatic capability, inhibited the ability of metastatic cells to cause platelet aggregation. However, another inhibitor of oligosaccharide processing, swainsonine, which inhibits mannosidase II activity and does not reduce metastasis, had no effect on platelet aggregation. These results show that the integrity of N-linked oligosaccharide structure of glycoproteins is an important feature of the ability of ras-transformed fibroblasts to activate platelets.     

Natural killer cell tumoricidal activity and deterioration of lung tumor metastasis in silicotic mice and stressed silicotic mice

Aim of this study is to explore the role of splenic NK cells in the lung metastasis in the silicotic mice. The number of metastatic foci increased 1.5-fold and 1.8-fold in the silicotic and stressed silicotic mice compared with the control mice. Treatment with an immunomodulator reduced the rate of tumor metastasis in silicotic mice with or without stresses, while their NK cell activity was normalized. Decrease of NK cell activity on the day of tumor inoculation but not on the post-inoculation days seems to be a major factor for predicting the extent of tumor metastasis in the silicotic and stressed silicotic mice.     

Inhibition by retinoids of platelet growth factor-dependent stimulation of DNA synthesis and cell division in density-arrested C3H 10T1/2 fibroblasts

The inhibition of neoplastic transformation by vitamin A and its natural and synthetic analogues, collectively called retinoids, is accomplished by an as yet unknown mechanism. In a recent report, the morphological transformation of carcinogen-treated C3H 10T1/2 fibroblasts to focus-forming transformed cells was shown to be a postconfluence event induced in density-arrested initiated cells by platelet growth factors in serum and was correlated with the mitogenic response of the preneoplastic cells to these polypeptides. The current study investigates the possibility that the inhibition of neoplastic transformation by retinoids is accomplished by blocking the mitogenic response of initiated cells to these growth factors. The results demonstrate that the stimulation by serum of DNA synthesis and cell division in normal and carcinogen-treated C3H 10T 1/2 fibroblasts after density-dependent growth arrest is inhibited in a dose-dependent manner by retinyl acetate, all-trans retinoic acid, and 4-hydroxyphenyl-retinamide over the same dose range and to the same extent that neoplastic transformation is inhibited by these retinoids. Cellular mitogenic processes sensitive to retinoid inhibition were shown to be induced specifically by platelet growth factors, rather than plasma growth factors, to occur within approximately 2 h of growth factor treatment, and to be common to cell division stimulated in density-arrested normal and initiated cells by highly purified platelet-derived or epidermal growth factor. These data suggest that the inhibition of neoplastic transformation by retinoids is accomplished by blocking the G0 to G1 transition in the mitotic response of initiated cells to platelet growth factors which act as endogenous promoters of transformation.     

Induction by growth factors from platelets of the focus-forming transformed phenotype in carcinogen-treated C3H /10T1 /2 fibroblasts

Wounding of mouse skin promotes tumor formation as effectivelyas 12-0-tetradecanoylphorbol-13-acetate(TPA). Like wounding,TPA stimulates the release of growth factors from plateletsand the leakage of plasma from the capillary circulation. Thus,initiated cells in TPA-treated skin are also exposed to thecomponents of whole blood-derived serum. It is possible thatserum factors play an important role in the multi-step processof neoplastic transformation. In this study, we evaluate thesignificance of serum components derived from plasma and plateletsin the neoplastic transformation of C3H /10T1 /2 mouse fibroblastsexposed to methylcholanthrene. Carcinogen-treated cultures grownto confluence in medium containing 5 % whole blood-derived serum(FBS), but maintained for 5 weeks after confluence in plasma-derivedserum (PDS), which lacks the platelet components found in wholeblood-derived serum, failed to produce transformed foci. Theaddition of an aqueous extract of platelets to PDS induced theformation of transformed foci with an efficiency comparableto FBS and proportional to the amount and mitogenic activityof the platelet extract. The growth of newly transformed cellswas not inhibited by the absence of platelet factors in theculture medium. The loss of densitydependent growth controlrequired at least 3 – 4 weeks of postconfluence exposureto serum factors derived from platelets. The data suggest thatplatelet factors induce the conversion of a carcinogen-initiatedcell to a focus-forming transformed cell. We demonstrate thatplatelet-derived growth factor (PDGF) is essential for the inductionof the focus-forming phenotype in initiated cells and that PDGFacts co-operatively with the platelet-derived type-ßtransforming growth factor and EGF-like growth factor to inducethis transformed phenotype. These growth factors may be actingas endogenous promoters of neoplastic transformation of chemicallyinitiated C3H /10T1 /2 mouse fibroblasts.     

早期结外NK/T细胞淋巴瘤的研究进展

结外NK/T细胞淋巴瘤(ENKTCL)是异质性强、高度侵袭性的非霍奇金淋巴瘤。尽管大多数ENKTCL患者诊断时为早期,但由于分期的局限性,预后差异较大。放疗和化疗是早期ENKTCL患者的一线治疗方法,但放疗、化疗模式及治疗时序仍存在争议。随着临床分期及预后模型的不断更新,早期ENKTCL患者倾向于接受风险适应性分层治疗...     

Effects of promoters on DNA synthesis in C3H/10T1/2 mouse fibroblasts

The synthesis of DNA has been studied by autoradiography and by measurements of tritiated thymidine ([3H]TdR) incorporation in cultured C3H/10T1/2 mouse embryo fibroblasts. The cells were first treated with 3-methylcholanthrene as an initiator and then with promoters according to schedules that produce oncogenic transformation. The levels of 3-methylcholanthrene used did not affect the growth or [3H]TdR incorporation of the cells. Treatment during the log phase of growth with 12-O-tetradecanoyl-phorbol-13-acetate, phorbol didecanoate, or 4alpha-phorbol didecanoate produced a transient inhibition of [3H]TdR incorporation with the maximum at 12 hr after treatment. This resulted in a temporary delay of growth followed by recovery of the normal cell-doubling time. Phorbol did not produce these effects, suggesting that the inhibition of DNA synthesis is associated with the process of promotion. Although treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate during stationary phase resulted in a 2- to 3-fold stimulation of [3H]TdR incorporation, multiple treatments spanning log and stationary phases were found to be necessary for promotion.     

Augmentation of postconfluence growth arrest of 10T1/2 fibroblasts by endogenous cyclic adenosine 3':5'-monophosphate

We have previously demonstrated that, by elevating intracellular adenosine 3':5'-cyclic monophosphate (cAMP) levels by inhibition of cAMP phosphodiesterase with Ro 20-1724 or by forskolin stimulation of adenylcyclase, the growth of neoplastically transformed 10T1/2 fibroblasts could be inhibited when these cells were in contact with growth-inhibited nontransformed 10T1/2 cells (J. S. Bertram and M. B. Faletto, Cancer Res., 45: 1946-1952, 1985) and furthermore that the extent of this growth inhibition correlated strongly with the degree of junctional communication between the two cell types (P.P. Mehta et al., Cell, 44: 187-196, 1986). To determine if these treatments enhance the degree of growth control of the nontransformed 10T1/2 cells, cultures were exposed to varying concentrations of Ro 20-1724 and/or forskolin. Drug treatment caused no significant effects on growth rate or cell spreading when cells were treated during logarithmic growth phase; however, major reductions of up to 70% in confluent saturation density and concomitant increases in cell spreading occurred in cultures making extensive cell/cell contacts. Decreases in saturation density correlated strongly with induced elevations of both intra- and extracellular cAMP concentrations. These effects could not be duplicated by the addition of exogenous cAMP agonists 8-bromo-cAMP and/or dibutyryl-cAMP. Two-dimensional electrophoresis of phosphate-labeled proteins revealed that forskolin treatment induced a quantitatively and qualitatively different phosphorylation profile than did 8-bromo-cAMP. Both basal and drug-induced intracellular cAMP levels fell as cells progressed from logarithmic to confluent growth state, implying that cells become sensitized to cAMP by the attainment of extensive cell/cell contacts. It is suggested that the drug-induced elevations of endogenously synthesized cAMP are accentuating a physiological role of cAMP on the postconfluent growth arrest of murine fibroblasts. The requirements for cell/cell contact and the known increased junctional communication induced by cAMP furthermore suggest that cAMP is enhancing the junctional transfer of a growth-inhibiting regulatory molecule. A likely candidate is cAMP itself.     

Natural killer T cells from interleukin-4-deficient mice are defective in early interferon-gamma production in response to alpha-galactosylceramide

Discovery of the natural killer (NK) T cell-specific ligand, alpha-galactosylceramide (alpha-GalCer) has enabled us to investigate the functional regulation of NKT cells. However, the detailed mechanism of cytokine production by NKT cells remains to be elucidated. Here we evaluated the role of interleukin (IL)-4 in the production of interferon (IFN)-gamma from NKT cells using IL-4-deficient C57BL/6 mice (IL-4(-/-) mice). Administration of alpha-GalCer into wild-type C57BL/6 mice caused the production of both IFN-gamma and IL-4 in serum or cytoplasm within 4 h of the injection. Unexpectedly, however, IL-4(-/-) mice-derived NKT cells did not produce any IFN-gamma at early phase after primary stimulation with alpha-GalCer. Because NKT cells from IL-4(-/-) mice produced IFN-gamma when they were stimulated secondarily with alpha-GalCer in vitro for 72 h, NKT cells from IL-4(-/-) mice were not completely genetically deficient for IFN-gamma production. To elucidate which cells, NKT cells or dendritic cells (DC), were responsible for the deficiency in IFN-gamma production in IL-4(-/-) mice, we carried out an add-back experiment using purified NKT cells and DC, which were prepared from either wild-type mice or IL-4(-/-) mice. NKT cells from wild-type mice produced IFN-gamma when they were cocultured with DC prepared from either wild-type or IL-4(-/-) mice, whereas NKT cells from IL-4(-/-) mice did not produce IFN-gamma by coculturing with DC from either wild-type or IL-4(-/-) mice. These results indicate that NKT cells, not DC, were responsible for the deficiency in IFN-gamma production in IL-4(-/-) mice. Thus, IL-4 is required for the activation of NKT cells to produce IFN-gamma in response to alpha-GalCer.     

Effect of growth state, tumor promoters, and transformation upon intercellular communication between C3H/10T1/2 murine fibroblasts

Diminished intercellular communication has been associated withheightened sensitivity of cultured cells to morphological transformationand enhancement of transformation by tumor promoters. Microinjectionof Lucifer yellow dye was employed to evaluate intercellularcommunication between transformable C3H/10T1/2 murine fibroblastsunder a variety of culture conditions. Intercellular communicationassayed by dye transfer from injected cells to surrounding cellsin contact occurred in logarithmically growing cultures, declinedto very low levels as confluence was attained, and then resumedupon the formation of mature confluent monolayers. Dye-transfernetworks of 50 or more cells resulted from Injection of singlemonolayer cells. Freeze-fracture electron microscopy confirmedthe presence of gap junction structures in confluent cultures.Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidineand/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxindid not inhibit intercellular communication between C3H/10T1/2cells during 6-week transformation experiments. The tumor promoter12-O-tetradecanoylphorbol-13-acetate produced a transient inhibitionof dye-coupling upon first introduction to cultures and prolongedthe period of diminished dye-coupling at the attainment of confluence,but did not inhibit subsequent interactions between monolayercells. A simple relationship thus could not be established betweenlevels of dye-coupling within monolayers and focus formationevents. Curiously, although the cells of foci in early phasesof development did not exhibit dye-transfer capacity, dye-couplingwas observed in mass cultures of most transformed cell linescloned from foci. Co-cultivation of communication-competenttransformed cells with nontransformed cells to produce reconstructedfoci generally resulted in a cessation of dye-transfer by transformedcells. An often reversible loss of communication competencethus accompanies the growth of transformed C3H/10T1/2 cellsas foci and may constitute an adaptive response which facilitatesfocus growth in the presence of intercellular communicationbetween monolayer cells.     

Natural killer cell lymphoma/leukemia: pathology and treatment

Malignancies arising from cells of putative natural killer (NK) cell origin have increasingly been recognized as distinct clinicopathological entities. These malignancies are marked by tumour cells with NK cell characteristics, including the immunophenotype of CD2⁺, surface CD3⁻, cytoplasmic CD3ϵ+, CD7±, and CD56+, and the genotype of germline T cell receptor gene. A consistent association with monoclonal Epstein–Barr virus infection in the tumour cell has been observed. These tumours are now regarded as putative NK cell lymphoma/leukemia. Pathologically, tumour cells show variable cytological appearances, with frequent angiocentricity and angioinvasion, associated with zonal necrosis. Clinically, most cases occur in the nasal area and upper aerodigestive tract. However, occurrence in non-nasal sites such as the skin, gastrointestinal tract and testis is also observed. A particularly aggressive form of NK lymphoma/leukemia presents fulminantly as disseminated disease sometimes with a leukemic phase. All types of NK lymphoma/leukemia have an extremely poor prognosis with a median survival of less than a year. New modalities of treatment, including the use of high dose chemotherapy and stem cell rescue may be needed to improve treatment outcome. © 1997 John Wiley & Sons, Ltd.     

Comparable growth regulation of five human tumor cell lines by neonatal human lung fibroblasts in semisolid culture media

Cellular growth interactions were studied between neonatal human lung fibroblasts (NLF-13) and human tumor lines derived from carcinomas of the prostate (PC-3, DU145), bladder (J82), and endometrium (HEC-1A) and from a glioma (Hs 683t). NLF-13 were interacted with tumor cells in soft agar or agarose media using two experimental protocols. In one system, NLF-13 cells were grown as anchored monolayers proliferating under the tumor cell layer. In the second, NLF-13 were embedded directly (nonanchored) into the agar or agarose layer with the tumor cells. The results from both interaction systems were similar for all five tumor lines. Anchored NLF-13 caused a dose-dependent inhibition of tumor growth, whereas nonanchored cells produced a dose-dependent growth stimulation. A time exposure experiment indicated that tumor stimulation and inhibition were biphasic responses to NLF-13. It was concluded that low concentrations of a diffusible NLF-13 product(s) accelerated tumor growth, whereas high concentrations were inhibitory. Further, the production of the active NLF-13 substance(s) was positively correlated with NLF-13 growth rate. Tumor cell inhibition was irreversible after a 5-day exposure to proliferating NLF-13 cells. Another line of normal neonatal human lung fibroblasts (NLF-147) showed inhibitory properties similar to those described for NLF-13. However, preliminary studies with fibroblasts from the skin of a Down's syndrome neonate (DS-172) and from a human kidney tumor (KTF-130) have shown both these fibroblast types to have a reduced ability to inhibit tumor cell cultures (J82) compared to the neonatal lung fibroblasts (NLF-13 and NLF-147).     

Initiation of C3H/10T1/2 cell transformation by N-methyl-N''-nitro-N-nitrosoguanidine and aflatoxin B1

The utility of C3H/10T1/2 mouse embryo fibroblasts for the detectionof carcinogenic substances has been limited by their apparentinsensitivity to the oncogenic effects of direct-acting alkylatingagents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) andprocarcinogens such as aflatoxin B₁ (AfB₁). Because the processof C3H/10T1/2 transformation can be observed to proceed throughdiscrete stages of initiation and promotion, we have consideredthe possibility that MNNG and AfB₁ may only initiate C3H/10T1/2transformation. Treatment of asynchronous C3H/10T1/2 cells withMNNG or AfB₁ alone generally produced few transformed foci.If MNNG or AfB₁ treatment was followed by the exposure of cellsto the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate(TPA), numerous transformed foci were produced. Phorbol didnot enhance transformation by either substance. MNNG and AfB₁thus appear to be initiating agents for transformation. TPAalso enhanced the transformation of C3H/10T1/2 cells by lowdoses of 3-methylcholanthrene (3-MCA), but transformation byhigh concentrations of 3-MCA was inhibited by the presence ofTPA. These studies suggest that the sensitivity of the C3H/10T1/2transformation system to potential carcinogens can be dramaticallyheightened if the bioassay is conducted in the presence andabsence of TPA.     

The role of nitric oxide in neoplastic transformation of C3H 10T1/2 embryonic fibroblasts

Nitric oxide synthase inhibitors block the neoplastic transformation of C3H 10T1/2 cells in vitro. Evidence presented herein suggests that they mediate their effects early in the carcinogenic process as brief treatment with the NOS inhibitor aminoguanidine (AG) during log phase cell growth (initiation phase) is sufficient to block foci formation. In contrast, treatment initiated after formation of a confluent monolayer was associated with diminished protection, while treatment commencing late in the promotional phase had no protective effect and appeared to enhance the number and stage of foci observed. These findings suggest that while AG treatment can inhibit transformation during the early promotional phase, it most effectively inhibits transformation during the initiation phase. In general AG enhanced growth of both normal and tumor cells, suggesting that effects on growth were unrelated to its anti-transformation properties, however, these effects could be related to the effect on tumor cell stage noted above. Although induction of inducible nitric oxide synthase (iNOS) by treatment with LI during the last 2 weeks of the assay was associated with enhanced transformation, the efficacy of AG in protecting against transformation was not clearly associated with substantial reductions in NO synthesis. The data suggest that AG inhibits transformation early in the transformation process independently of iNOS inhibition and that AG may have deleterious effects late in the process, possibly through stimulation of tumor cell growth.     

Natural killer and lymphokine activated killer cell functions in Hodgkin's disease

We report the natural killer (NK) and lymphokine activated killer (LAK) cell activities in peripheral blood lymphocytes (PBL) from untreated patients with Hodgkin's disease (HD) and from healthy donors. The frequency of LAK cell precursors was also studied using limiting dilution analysis (LDA). About 75% of the HD patients had normal NK activity. There was a higher percentage of low NK responders (mean percent NK activity of healthy donors--2 SD) in patients with lymphocyte depletion histologic grade of the disease and those who were in clinical stage IV, suggesting a correlation of decrease in NK activity with poor prognosis. We found efficient LAK activity against the NK-sensitive K562 cells and NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumour targets in both low and normal NK responder HD patients, irrespective of the histopathological grade and clinical stage of the disease. In concordance with their good LAK cell activity, HD patients showed a frequency distribution of LAK cell progenitors in the PBL comparable to that of healthy donors.     

Natural killer cell lymphoma/leukemia with homozygous loss of p27/kip1.

We describe a case of natural killer (NK) cell lymphoma/leukemia with only an interstitial deletion in the short arm of chromosome 12 as the primary event. Fluorescence in situ hybridization revealed that the ETV6 locus (12p13) and subtelomeric sequences are not deleted in the process. The p27/kip1 locus (12p12-13), a candidate tumor suppressor gene, was deleted on the abnormal chromosome. Sequence analysis detected an adenine nucleotide deletion in the third codon of exon 1 leading to frameshift and premature termination at codon 41 of the retained copy of p27/kip1. To the best of our knowledge, this is the first report in literature on a NK cell lymphoma/leukemia with complete loss of p27/kip1.     

Quantitative neoplastic transformation of C3H/10T1/2 fibroblasts: dependence upon the size of the initiated cell colony at confluence

The efficiency of transformation of C3H/10T1/2 fibroblasts by chemical or physical carcinogens varies inversely with the seeding density of the cells. Colony size at confluence, accumulated cell generations, and epigenetic events independent of seeding density have been proposed to explain this variability. By controlling the total number and colony size (i.e., cells/colony) of initiated cells at confluence and their accumulated cell generations, we have determined that (a) the colony size of the initiated cells at confluence is directly related to the expression of the transformed phenotype, (b) the probability that an initiated cell will form a colony of transforming cells does not vary with the accumulation of large numbers of cell generations, and (c) the total number of initiated cells in a confluent culture does not determine the number of transformed foci formed. We have developed a mathematical representation which, when applied to our results and the published results from other laboratories, clearly describes the relationship between the size of the initiated cell colony at confluence and the expression of the transformed phenotype. These results have significant implications in the quantification of the transformation of C3H/10T1/2 cells by chemical and physical carcinogens.     

Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-beta1 heterozygous mice

We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 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-beta1 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 p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), 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 p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), 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 p15(Ink4b) and p27(Kip1) 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 p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 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.