Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin-ezrin-radixin family of cytoskeleton-associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Skin tumor-promoting activity of benzoyl peroxide, a widely used free radical-generating compound.

Benzoyl peroxide, a widely used free radical-generating compound, promoted both papillomas and carcinomas when it was topically applied to mice after 7,12-dimethylbenz[a]anthracene initiation. Benzoyl peroxide was inactive on the skin as a complete carcinogen or as a tumor initiator. A single topical application of benzoyl peroxide produced a marked epidermal hyperplasia and induced a large number of dark basal keratinocytes, effects similar to those produced by the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. Benzoyl peroxide, like other known tumor promoters, also inhibited metabolic cooperation (intercellular communication) in Chinese hamster cells. In view of these results caution should be recommended in the use of this and other free radical-generating compounds.

AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Cell cycle-regulated processing of HEF1 to multiple protein forms differentially targeted to multiple subcellular compartments.

HEF1, p130(Cas), and Efs/Sin constitute a family of multidomain docking proteins that have been implicated in coordinating the regulation of cell adhesion. Each of these proteins contains an SH3 domain, conferring association with focal adhesion kinase; a domain rich in SH2-binding sites, phosphorylated by or associating with a number of oncoproteins, including Abl, Crk, Fyn, and others; and a highly conserved carboxy-terminal domain. In this report, we show that the HEF1 protein is processed in a complex manner, with transfection of a single cDNA resulting in the generation of at least four protein species, p115(HEF1), p105(HEF1), p65(HEF1), and p55(HEF1). We show that p115(HEF1) and p105(HEF1) are different phosphorylation states of the full-length HEF1. p55(HEF1), however, encompasses only the amino-terminal end of the HEF1 coding sequence and arises via cleavage of full-length HEF1 at a caspase consensus site. We find that HEF1 proteins are abundantly expressed in epithelial cells derived from breast and lung tissue in addition to the lymphoid cells in which they have been predominantly studied to date. In MCF-7 cells, we find that expression of the endogenous HEF1 proteins is cell cycle regulated, with p105(HEF1) and p115(HEF1) being rapidly upregulated upon induction of cell growth, whereas p55(HEF1) is produced specifically at mitosis. While p105(HEF1) and p115(HEF1) are predominantly cytoplasmic and localize to focal adhesions, p55(HEF1) unexpectedly is shown to associate with the mitotic spindle. In support of a role at the spindle, two-hybrid library screening with HEF1 identifies the human homolog of the G2/M spindle-regulatory protein Dim1p as a specific interactor with a region of HEF1 encompassed in p55(HEF1). In sum, these data suggest that HEF1 may directly connect morphological control-related signals with cell cycle regulation and thus play a role in pathways leading to the progression of cancer.

Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.

High CD44 expression is associated with enhanced malignant potential in esophageal squamous cell carcinoma (ESCC), among the deadliest of all human carcinomas. Although alterations in autophagy and CD44 expression are associated with poor patient outcomes in various cancer types, the relationship between autophagy and cells with high CD44 expression remains incompletely understood. In transformed oesophageal keratinocytes, CD44Low-CD24High (CD44L) cells give rise to CD44High-CD24-/Low (CD44H) cells via epithelial-mesenchymal transition (EMT) in response to transforming growth factor (TGF)-ß. We couple patient samples and xenotransplantation studies with this tractable in vitro system of CD44L to CD44H cell conversion to investigate the functional role of autophagy in generation of cells with high CD44 expression. We report that high expression of the autophagy marker cleaved LC3 expression correlates with poor clinical outcome in ESCC. In ESCC xenograft tumours, pharmacological autophagy inhibition with chloroquine derivatives depletes cells with high CD44 expression while promoting oxidative stress. Autophagic flux impairment during EMT-mediated CD44L to CD44H cell conversion in vitro induces mitochondrial dysfunction, oxidative stress and cell death. During CD44H cell generation, transformed keratinocytes display evidence of mitophagy, including mitochondrial fragmentation, decreased mitochondrial content and mitochondrial translocation of Parkin, essential in mitophagy. RNA interference-mediated Parkin depletion attenuates CD44H cell generation. These data suggest that autophagy facilitates EMT-mediated CD44H generation via modulation of redox homeostasis and Parkin-dependent mitochondrial clearance. This is the first report to implicate mitophagy in regulation of tumour cells with high CD44 expression, representing a potential novel therapeutic avenue in cancers where EMT and CD44H cells have been implicated, including ESCC.

Differentiation of normal and cultured preneoplastic tracheal epithelial cells in rats: importance of epithelial mesenchymal interactions.

Changes in the dependence on mesenchymal tissues for survival and differentiation in inbred F344 female rats were investigated in tracheal epithelial cells exposed to 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). Fresh suspensions of normal tracheal epithelium or cultured preneoplastic cells were inoculated into isolated organ segments (trachea, esophagus, bladder, or small intestine) or into Dacron containers that were then implanted subdermally into isogenic recipients. At various times after cell inoculation and implantation, tissues were removed for histologic evaluation. Normal cells inoculated into frozen-thawed trachea, esophagus, bladder, and intestine yielded a regular mucociliary epithelium. Normal cell inocula did not, however, survive in tracheae previously heated (100 degrees C), fixed in ethanol, or digested with collagenase; nor did normal cells survive in Dacron containers unless tracheal fibroblasts plus epithelial cells were inoculated together. DMBA- and TPA-exposed cell populations with increased growth capacity in vitro survived and differentiated on all of the above substrates. Our observations were consistent with those of other investigators in that normal cell survival and differentiation depend to some extent on interaction with extracellular material(s) present in various organs. The essential elements were not supplied by subdermal fibroblasts alone. For survival and differentiation in vivo, preneoplastic cells appeared to have less stringent substrate requirements than did normal cells. Application of the described techniques to the study of changes occurring early in the development of neoplastic disease is discussed.

Modulation of growth, differentiation, and mucous glycoprotein synthesis by retinyl acetate in cloned carcinoma cell lines.

The ability of retinyl acetate to alter growth, differentiation, and synthesis of mucous glycoproteins in cell lines cloned from an adenocarcinoma (T-8) and a squamous cell carcinoma (1000 WT) was investigated with the use of F344 rats. Growth rate was inhibited approximately 25 and 50% in 6.6 x 10(-6) and 3.3 x 10(-5) M retinyl acetate, respectively. In both cell lines. Cell line T-8 grew mainly as a monolayer, whereas cell line 1000 WT grew as a stratified epithelium. In the presence of both concentrations of retinyl acetate, this stratification was decreased and cells became enlarged and more cuboidal. Retinyl acetate induced the formation of numerous vacuoles and periodic acid-silver methenamine-positive granules in both T-8 and 1000 WT cells. The granules appeared with and without dense cores in electron micrographs. Golgi hypertrophy and increased numbers of microvilli were also evident. After T-8 cells were cultured for 7 days in 6.6 x 10(-6) or 3.3 x 10(-5) M retinyl acetate, [3H]glucosamine incorporation increased 133- to 147-fold and [14C]serine incorporation increased twelvefold to twentyfold in the high-molecular-weight mucous glycoprotein fraction (peak A) from the cell cytosol. In 1000 WT cells, [3H]glucosamine incorporation creased only 4.2- to 7.5-fold, and [14C]serine incorporation increased only 2.6- to 4.6-fold under the same culture conditions. A similar difference in the amount of stimulation was seen for peak A isolated from the secretions. Thus T-8 cells showed a marked increase in the synthesis and secretion of mucins, whereas 1000 WT cells showed a comparatively small but significant increase.

Lectin-binding sites in preneoplastic and neoplastic lesions of human and rodent respiratory tracts.

The localization of fluorescein-labeled lectins, i.e., concanavalin A (Con A), Ricinus communis-120 (RCA), and wheat-germ agglutinin (WGA), were studied histologically in F344 rat epithelial lesions produced in the course of chemical carcinogenesis. WGA could not be demonstrated in these lesions. Although all lesions showed positive-binding sites when high concentrations of either Con A or RCA were used, a dilution study showed that the epithelial lesions had different affinities for lectins. With both Con A and RCA, dysplastic and neoplastic lesions showed the strongest intensity of fluorescence and squamous metaplasia showed the weakest. Normal and hyperplastic epithelia showed intermediate intensity. In the dilution study, RCA showed eight times more affinity and Con A showed two times more affinity for dysplastic and neoplastic epithelia than for normal or hyperplastic epithelium. Similar affinity patterns were observed in human lesions and tumors. With Con A, 58% of tumors showed much stronger fluorescence than did normal epithelium, and 44% of the tumors showed positive fluorescence with RCA. Although both lectins exhibited a stronger affinity for all the dysplastic-neoplastic lesions than for normal or hyperplastic epithelium, RCA proved to be the most adequate marker for preneoplastic lesions.

Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells.

BACKGROUND: Multidrug resistance-associated protein (MRP) 1 and canalicular multispecific organic anion transporter (cMOAT or MRP2) are adenosine triphosphate-binding cassette transporters that confer resistance to anticancer agents. In addition to these two transporters, there are at least four other human MRP subfamily members (MRP3 through MRP6). We and others reported previously that MRP3 is capable of conferring resistance to certain anticancer agents. In this study, we investigated whether MRP4 (MOAT-B), whose transcript accumulates to the highest levels in prostate tissue, has the capacity to confer drug resistance. METHODS: MRP4-transfected NIH3T3 cells were generated, and their drug sensitivity was analyzed. The subcellular localization of MRP4 was assessed by immunohistochemical analysis in transfected cells and in prostate tissue. Statistical tests were two-sided. RESULTS: MRP4 was detected as a 170-kd protein that was localized in the plasma membrane and cytoplasm of transfected cells. The MRP4 transfectants displayed 5.5-fold increased resistance to methotrexate in short-term drug-exposure assays (P=.022) and exhibited decreased cellular accumulation of this agent at 4 hours (P=.006) and 24 hours (P<.001). In continuous-exposure assays, however, the MRP4 transfectants did not display increased resistance for either methotrexate or natural product cytotoxic agents (anthracyclines, etoposide, vinca alkaloids, and paclitaxel [Taxol]). However, the transfectants did show increased resistance (2.3-fold) for the anti-acquired immunodeficiency syndrome nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) (P=.022) in continuous-exposure assays. Consistent with MRP4's plasma membrane localization in transfected cells, analysis of prostate tissue showed that MRP4 protein was localized primarily in the basolateral plasma membranes of tubuloacinar cells. CONCLUSIONS: These results indicate that MRP4 confers resistance to short-term methotrexate and continuous PMEA treatment. Given its structure, drug resistance profile and subcellular localization, MRP4 probably functions as an amphipathic anion efflux pump whose substrate range includes glutamate and phosphate conjugates.

Simultaneous appearance of keratin modifications and gamma-glutamyltransferase activity as indicators of tumor progression in mouse skin papillomas.

gamma-Glutamyltransferase (GGT), an enzyme not found in normal adult epidermis, was detected in most skin papillomas larger than 13 mm in diameter and in all squamous carcinomas induced by 7,12-dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol 13-acetate promotion in noninbred Sencar mice. Furthermore, these GGT-positive lesions were also characterized by a marked decrease or absence of high-molecular-weight components of epidermal keratin. Since these characteristics are common to both carcinomas and large papillomas but are practically undetectable in normal epidermis and small papillomas, GGT activity and lack of high-molecular-weight keratin components seem to be good indicators of tumor progression, i.e., from papilloma to squamous carcinoma.

Effect of carcinogen release rate on the incidence of preneoplastic and neoplastic lesions of the respiratory tract epithelium in rats.

Inbred F34 rat tracheal transplants were exposed to 7,12-dimethylbenz[a]anthracene (DMBA) delivered at different release rates for intraluminal pellets made of various matrices to study the effect of carcinogen dose rate on the induction of lesions in the epithelium. These matrices were beeswax, beeswax-stearyl alcohol, and beeswax-cholesterol. In addition, DMBA absorbed onto carbon particles was dispersed in beeswax-stearyl alcohol. The fastest release was obtained from beeswax pellets from which 99% of the carcinogen (198 micrograms) was released in 4 weeks, and the slowest release was from DMBA absorbed on carbon at a ratio of 1:9 from which only 56% (113 micrograms) was released in 16 weeks. Morphometry of histologic sections showed marked differences in the percentage of luminal surface covered by dysplastic-neoplastic epithelium (i.e., 7.5% in the tracheas exposed to the fastest releasing pellets and 46.3% in the tracheas exposed to the slowest releasing pellets). An inverse linear correlation was found between the cumulative amount of DMBA relased from the different pellet matrices of 2 weeks and the incidence of dysplastic plus neoplastic lesions of tracheal epithelium at 16 weeks. The results indicate that lower doses of carcinogen delivered slowly are more effective in producing dysplastic plus neoplastic lesions than hgher doses delivered rapidly.

Numerical variation of dark cells in normal and chemically induced hyperplastic epidermis with age of animal and efficiency of tumor promoter.

The percentages of dark keratinocytes was quantitatively assessed in normal epidermis of Sencar mice before and after birth and in adult epidermis after topical application of several compounds of varying promoting efficiency. The percentage of dark keratinocytes reached a maximum at the 19th day of gestation (approximately 40%) and fell abruptly after birth (approximately 3%). Old animals exhibited a very low number of dark basal cells (0.2%). After topical application of the weak promoters resiniferotoxin, anthralin, ethylphenylpropiolate, and 12-deoxyphorbol-13-2,4,6-decatrienoate, the percentage of dark cells in young adult epidermis did not differ markedly from that in control (acetone-treated) specimens. The strong first-stage promoters 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and calcium ionophore A 23187, as well as the strong complete promoter 12-deoxyphorbol-13-deoxyphorbol-13-decanoate, induced the appearance of large numbers of dark keratinocytes, in a percentage similar to that seen after 12-O-tetradecanoylphorbol-13-acetate application (approximately 20%). The similarities between the dark keratinocytes seen after topical application of 12-O-tetradecanoylphorbol-13-acetate or other strong promoters and the dark cells observed in the fetal epidermis before the onset of the adult type of epidermal keratinization indicate that potent and/or first stage tumor promoters can be identified by their ability to induce cells resembling fetal-type dedifferentiated keratinocytes.

Uptake of temozolomide in a rat glioma model in the presence and absence of the angiogenesis inhibitor TNP-470.

The angiogenic phenotype is associated with hyperpermeable capillaries. Through treatment with angiogenesis inhibitors capillary permeability may be reduced, and it can be anticipated that cytotoxic agents coadministered may be adversely affected. The current investigation examined this possibility for the combination of TNP-470, an angiogenesis inhibitor, and temozolomide (TMZ), a DNA-alkylating agent with demonstrated activity in brain tumors. TNP-470 (30 mg/kg) was given s.c. on days 6, 8, 10, 12, and 14 following s.c. implantation of rat C6 glioma cells in Sprague-Dawley rats. On the 15th day following tumor implantation, control (no TNP-470) and treated rats received 40 mg/kg of TMZ intraarterially. Prior to dosing, a linear microdialysis probe was placed in the tumor to collect interstitial fluid. Plasma and interstitial fluid samples were collected for 8 h and measured for TMZ by a high-performance liquid chromatography assay. Pharmacokinetic parameters for TMZ were calculated by noncompartmental methods. Total systemic clearance (39.8 +/- 7 versus 44.2 +/- 14 ml min(-1) kg(-1)) and volume of distribution (5.4 +/- 2 versus 5.2 +/- 0.8 L kg(-1)) were not significantly different in control and TNP-470-treated animals. However, the mean TMZ area under the interstitial fluid concentration-time curve was reduced by 25% in the TNP-470-treated group compared to the control (5450 +/- 1892 versus 4120 +/- 1790 micrograms min ml(-1); P < 0.05). It appears that TNP-470 caused this reduction in the tumor uptake of TMZ by its pharmacodynamic action on the tumor vasculature. Since combination regimens using angiogenesis inhibitors and cytotoxic drugs will be needed to determine how such combinations can be used effectively. The current animal model, which utilized tumor microdialysis, can serve as a model to further analyze combination chemotherapy.

Ultraviolet radiation-induced malignant skin melanoma in melanoma-susceptible transgenic mice.

A new mouse model of UV radiation-induced melanoma is described. Unlike previous models, melanoma induction requires only short-term irradiation, does not require the application of chemical carcinogens, and does not cause any other tumors. The model takes advantage of the fact that Tyr-SV40E (C57BL/6 strain) transgenic mice are all melanoma-susceptible, and that different inbred lines are susceptible to different extents. Four-day-old mice of a moderately susceptible line (line 9 transgenic homozygotes) were exposed for 20 min/day to 328 mJ/cm2 to UVB (280-320 nm wavelength, comprising 70% of the total irradiance) for up to 4 consecutive days. Melanocytic lesions resembling macules, nevi, or early melanomas gradually appeared in the irradiated mice that were not seen in unirradiated transgenic controls of similar age. To afford ongoing observation beyond the short life span of line 9 homozygous mice, skin samples containing a total of 26 selected lesions were grafted at 20 weeks after UV radiation to longer-lived unirradiated hosts of transgenic line 12, in which melanoma susceptibility is low. Ten lesions in the grafts became melanomas; all melanomas had ulcerated and two had metastasized. At the stages examined, all the tumors were deeply melanotic. The remaining 16 lesions were still indolent when the experiment was terminated at 57 weeks post-UV radiation. The present protocol lends itself to variations in the choice of transgenic line, the age of the treated mice, and the intensity and duration of ultraviolet light; appropriate combinations of these variables would be expected to yield melanomas in relatively long-lived transgenic mice without skin grafting. The new model provides an opportunity to determine the melanoma action spectrum, to characterize at the molecular level the melanomas induced by ultraviolet light in comparison with those of other origins, and to investigate in vivo the photoprotective role of melanin.

A morphometric study of dedifferentiated and involutional dark keratinocytes in 12-O-tetradecanoylphorbol-13-acetate-treated mouse epidermis.

The epidermis of mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated using the method of stereological cytology. These procedures were carried out in order to quantify the morphological changes taking place in basal cells, the presumed target cells of TPA in the epidermis. The basal layer of the treated epidermis was composed of TPA-induced dark basal keratinocytes (DC) and clear basal keratinocytes. TPA-induced DC could be divided into two types. Type 1 was characterized by a relatively high volume fraction of ribosome and nonbundled filaments and by a low volume fraction of bundled filaments. These characteristics of type 1 DC were relatively constant over the observation period. Type II DC were characterized by a high volume density of mitochrondria , bundled filaments, and cytoplasmic vacuoles. This type of DC was seldom seen in normal epidermis but constituted approximately 20% of the dark cells in the epidermis 24 and 48 hr after treatment with TPA. In contrast, clear basal keratinocytes exhibited time-dependent changes such as an increase of the relative volume of nuclei; an increase in the volume density of mitochondria, nonbundled filaments, and ribosomes; and a decrease in the volume density of bundled filaments. Both the qualitative and quantitative analyses of TPA-treated and fetal epidermis suggest a close similarity between type 1 DC and DC seen in normal fetal epidermis, thus supporting the view that type 1 DC are poorly differentiated cells or dedifferentiated cells, different from type II or involutional DC.

Sequential study of gamma-glutamyltransferase during complete and two-stage mouse skin carcinogenesis.

The histochemical pattern of gamma-glutamyltransferase (GGT) was studied in benign and malignant tumors produced by two different experimental protocols, two-stage carcinogenesis and complete carcinogenesis. Six percent of all papillomas produced by two-stage carcinogenesis were GGT positive, whereas 14% of benign tumors produced by complete carcinogenesis exhibited GGT-positive areas. The incidence of GGT-positive papillomas in the two-step carcinogenesis protocol increased up to wk 28 of treatment. After 32 wk, the incidence decreased abruptly, coinciding with an abrupt increase in the incidence of squamous cell carcinomas. On the other hand, the incidence of GGT-positive benign tumors produced during the course of complete carcinogenesis increased gradually up to wk 32 of treatment, coinciding with the increased incidence of squamous cell carcinomas. The incidence of GGT-positive keratoacanthomas and GGT-positive papillomas produced with the complete carcinogenesis protocol exhibited different patterns, suggesting different histogenesis and biological behavior of these two types of tumors. In addition, the labeling index of GGT-positive areas was lower (17 +/- 3%) than that of the GGT-negative areas (41 +/- 0.18%) of the same papillomas, indicating that the presence of GGT may be related to abnormal keratocyte differentiation rather than to proliferative changes.

Enhanced acute lung damage in mice following administration of 1,3-bis(2-chloroethyl)-1-nitrosourea.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), also known as carmustine, is a lipid soluble anticancer drug which produces pulmonary fibrosis in up to 30% of the patients who receive this drug. The major risk factor for this disorder is preexisting lung damage. Animal models of this interaction have not been reported previously. A diffuse alveolar lesion was produced in male BALB/c mice by the administration of butylated hydroxytoluene (BHT). Total lung hydroxyproline levels, an index of fibrosis, were not increased in mice 21 days after single doses of BHT or BCNU. Total lung DNA synthesis, an index of pulmonary damage, was slightly increased after 15 and 18 days in rats and mice treated with BCNU (15 and 35 mg/kg, respectively). This suggested that a single dose of BCNU had only a minimal toxic effect on lung tissue. Combined treatments in mice given BHT (350 or 400 mg/kg), followed on Day 1 by BCNU (35 mg/kg), resulted in the deposition of significantly more hydroxyproline than with either agent alone. This enhancement was not seen following lower doses of BHT and was diminished when the dose of BCNU was decreased. Delaying the administration of BCNU (35 mg/kg) until Day 3 or 5 eliminated increases in hydroxyproline content, but not histological evidence of enhanced lung damage. Additional histological analyses confirmed the presence of an increased fibrotic reaction, especially when high doses of BCNU were administered 1 day after BHT (400 mg/kg). Most of the lungs were totally consolidated with numerous hyperactive fibroblasts and a large number of giant type II cells with atypical nuclei. These effects may be related to the ability of BCNU to inhibit pulmonary glutathione reductase activity and the increased DNA synthesis normally seen after BHT. These data show that BCNU treatment can enhance BHT-induced lung damage resulting in a fibrotic lesion similar to that seen in some human patients. This effect is dependent on the extent of the initial lung lesion as well as the time when BCNU is administered and may represent an animal model of the primary risk factor for the development of pulmonary fibrosis in human patients receiving this drug.

Progression of squamous carcinoma cells to spindle carcinomas of mouse skin is associated with an imbalance of H-ras alleles on chromosome 7.

Analysis of benign and malignant mouse skin tumors had previously shown that amplification of a mutant H-ras allele or loss of the normal allele was generally seen only in high grade or spindle cell tumors. The normal:mutant ras gene dosage has been studied directly by polymerase chain reaction amplification of DNA derived from paraffin sections of carcinomas of defined histological types. Some tumors had virtually no signal corresponding to the normal allele and these were invariably spindle cell carcinomas. In four cases where both squamous and spindle cell components could be identified within the same tumor the spindle cell component had a higher mutant:normal gene ratio. Additional experiments on cell lines derived from squamous or spindle cell tumors have demonstrated a good correlation between the ratio of normal:mutant ras and the degree of invasiveness of the cells in in vitro assays.

Expression of p21 is not required for senescence of human fibroblasts.

Senescence and immortalization have been studied in skin fibroblasts derived from two individuals with the Li-Fraumeni syndrome. These cells inherit one wild-type and one mutant p53 allele and lose the former during culture. Despite this loss, cultures of Li-Fraumeni syndrome cells progressed normally from early passage to replicative senescence. Senescent cells also expressed barely detectable levels of p21 mRNA, and, in marked contrast to normal cultured cells, levels of p21 expression decreased during in vitro aging. Further maintenance for up to 10 months of post-mitotic cultures has led to the isolation of cells with an extended lifespan. Four potentially immortal cultures have continued to proliferate, and two have completed more than 110 population doublings. These results indicate that p53 and p21 are not required for replicative senescence in human fibroblasts. However, their inactivation may enhance the probability of spontaneous immortalization.