Traditional preparations of kava (Piper methysticum) inhibit the growth of human colon cancer cells in vitro.

BACKGROUND: Epidemiological studies indicate there is low incidence of colon cancer in the South Pacific islands, including Fiji, West Samoa, and Vanuatu. Cancer incidence has been shown to be inversely associated with kava (Piper methysticum G. Forst.) ingestion. Hypothesis/Purpose: Kava prepared traditionally will inhibit the growth of human cancer cells. This investigation entails preparation and analysis of kava extracts and study of the growth inhibitory activity of the extracts, alone and combined with hibiscus. STUDY DESIGN: We will prepare kava as in Micronesia - as a water extract, high in particulate content, alone or combined with sea hibiscus (Hibiscus tiliaceus L.) - and examine the components and growth inhibitory activity. METHODS: We obtained ground kava prepared in the traditional way from lateral roots and sea hibiscus mucilage and sap from different sources in Micronesia, and prepared water extracts (unfiltered, as well as filtered, since in traditional use the kava beverage contains a high particulate content) and partitions. We used the MTT assay to determine the growth inhibitory activity of the preparations on colon and breast cancer cells and nonmalignant intestinal epithelial cells. LC-MS analysis was used to examine the components of the kava and sea hibiscus extracts and partitions. RESULTS: Traditional preparations of kava inhibit the growth of breast and colon cancer cells. Among the kava preparations, the order of decreasing activity was Fiji(2), Fiji(1), Hawaii; the unfiltered preparations from Fiji were more active than the filtered. Phytochemical analysis indicated that filtering reduced most kavalactone and chalcone content. For example, for Fiji(2), the ratio of dihydromethysticin in filtered/unfiltered kava was 0.01. Thus, for the extracts from Fiji, growth inhibitory activity correlates with the content of these compounds. Unfiltered and filtered kava from Fiji(1) were more active on malignant than nonmalignant intestinal epithelial cells. Since kava is prepared in Micronesia by squeezing the extract through sea hibiscus bark, we assayed the growth inhibitory activity of combinations of kava and sea hibiscus sap and found that sea hibiscus enhanced the growth inhibitory effect of kava. CONCLUSION: Our results show that traditional kava, alone or combined with sea hibiscus, displays activity against human cancer cells and indicate it will be worthwhile to develop and further analyze these preparations to prevent and treat colon and other cancers. Our findings suggest it is important to examine the activity of plants in the form that people consume them.

A Fraxinus excelsior L. seeds/fruits extract benefits glucose homeostasis and adiposity related markers in elderly overweight/obese subjects: a longitudinal, randomized, crossover, double-blind, placebo-controlled nutritional intervention study.

PURPOSE: The aim of this study was to investigate the potential benefits of an extract obtained from seeds/fruits of an Oleaceae (Fraxinus excelsior L.) on glucose homeostasis and associated metabolic markers in non-diabetic overweight/obese subjects. MATERIALS AND METHODS: This study was performed in 22 participants (50-80 years-old; BMI 31.0 kg/m(2)). The design was a longitudinal, randomized, crossover, double-blind, placebo-controlled 7-week nutritional intervention. The participants received daily 3 capsules each containing either 333 mg of an extract from Fraxinus excelsior L. seeds (Glucevia(®)) or placebo capsules (control) in a random order for 3 weeks with 1 week of washout between treatments. Moreover, they followed a balanced covert energy-restricted diet (-15% energy). All variables were measured at the beginning and at the end of each period. RESULTS: Compared to baseline, the administration of 1 g of Glucevia(®) for 3 weeks resulted in significantly lower incremental glucose area under the curve (-28.2%; p<0.01), and significantly lower 2 h blood glucose values (-14%; p<0.01) following an oral glucose tolerance test. No significant changes were found in the control group (-7.9% AUC, -1.6% 2h blood glucose). Furthermore, significant differences were found between responses in the control and Glucevia(®) groups with respect to serum fructosamine and plasma glucagon levels (p<0.01 and p<0.05, respectively). Interestingly, administration of Glucevia(®) significantly increased the adiponectin:leptin ratio (p<0.05) and decreased fat mass (p<0.01) compared to control (p<0.05). CONCLUSION: The administration of an extract from Fraxinus excelsior L. seeds/fruits in combination with a moderate hypocaloric diet may be beneficial in metabolic disturbances linked to impaired glucose tolerance, obesity, insulin resistance and inflammatory status, specifically in older adults.

Active compounds and distinctive sensory features provided by American ginseng (Panax quinquefolius L.) extract in a new functional milk beverage.

American ginseng (Panax quinquefolius L.) has recognized neurocognitive effects, and a ginsenoside-rich extract of the root of the plant has been shown to improve cognitive functions in young adults. This study aimed at assessing the chemical and sensory profiles of a UHT-treated, low-lactose functional milk containing American ginseng. Individual ginsenosides in the milk were analyzed by HPLC. Descriptive sensory analysis was performed by a trained panel to quantitatively document sensory changes resulting from the addition of ginseng and the UHT process on flavored and unflavored milks. Consumer acceptance of the product was also investigated. Total ginsenoside content in the UHT-treated milk enriched with the ginseng extract after UHT process treatment was 7.52 mg/100 g of milk, corresponding to a recovery of 67.6% compared with the content in the unprocessed extract. The intake of 150 to 300 mL of this ginseng-enriched milk provides the amount of total ginsenosides (11.5 to 23 mg) necessary to improve cognitive function after its consumption. Both the presence of ginsenosides and their thermal treatment affected some sensory properties of the milk, most notably an increase in bitterness and metallic taste, the appearance of a brownish color, and a decrease in milky flavor. Levels of brown color, bitterness, and metallic taste were highest in the industrially processed ginseng-enriched milk. The bitterness attributable to ginseng extract was reduced by addition of vanilla flavor and sucralose. A consumer exploratory study revealed that a niche of consumers exists who are willing to consume this type of product.

Comparison of different approaches for assessment of HER2 expression on protein and mRNA level: prediction of chemotherapy response in the neoadjuvant GeparTrio trial (NCT00544765).

Human epidermal growth factor receptor 2 (HER2) testing is an essential part of pathological assessment in breast cancer patients, as HER2 provides not only prognostic but also predictive information on response to targeted therapy. So far, HER2 test accuracy of immunohistochemistry/in situ-hybridization techniques is still under debate, and more reliable and robust technologies are needed. To address this issue and to evaluate the predictive value of HER2 on chemotherapy, we investigated a cohort of 278 patients from the GeparTrio trial, a prospective neoadjuvant anthracycline/taxane-based multicenter study. In the GeparTrio trial, patients were not treated with any anti-HER2 therapy, as this was not standard therapy at this time. The HER2 status was analyzed by three different approaches: local and central evaluation using immunohistochemistry combined with in situ-hybridization as well as evaluation of HER2 mRNA expression using kinetic RT-PCR from formalin-fixed, paraffin-embedded (FFPE) tissue samples using a predefined cutoff. HER2 overexpression/amplification was observed in 37.3% (91/244) and 17.9% (41/229) of the informative samples in the local and central evaluations, respectively. Positive HER2 mRNA levels were found in 19.8% (55/278). We observed a highly significant correlation between central HER2 expression and HER2 status measured by kinetic RT-PCR (r = 0.856, P < 0.0001) and an overall agreement of 95.6% (κ statistic, 0.862, CI 0.77-0.94). Further, central HER2 as well as HER2 mRNA expression were predictors for a pathological complete response after neoadjuvant anthracycline/taxane-based primary chemotherapy in a univariate binary logistic regression analysis (OR 3.29, P = 0.002; OR 2.65, P = 0.004). The predictive value could be confirmed for the central HER2 status by multivariate analysis (OR 3.04, P = 0.027). The locally assessed HER2 status was not predictive of response to chemotherapy. Our results suggest that standardized methods are preferable for evaluation of HER2 status. The kinetic RT-PCR from FFPE tissue might be an additional approach for assessment of this important prognostic and predictive parameter but has to be confirmed by other studies.

Classification of man-made vitreous fibers: Comments on the revaluation by an IARC working group.

In 2001, an IARC working group revaluated the carcinogenic risks of man-made vitreous fibers (MMVF). Compared with the IARC evaluation in 1987, the overall evaluations of insulation glass wool, rock (stone) wool, and slag wool were changed from Group 2B to Group 3. These changes ensued from an alteration in the evidence for cancer in humans and in experimental animals: Instead of "sufficient," the evidence for cancer in experimental animals is now looked upon as "limited" if there is a carcinogenic response after intraperitoneal injection but not after recently conducted inhalation experiments. For these studies, it is argued that they did properly address the technological limitations of earlier inhalation experiments. For Maxim and McConnell [Maxim L.D., McConnell E.E., 2001. Interspecies comparisons of the toxicity of asbestos and synthetic vitreous fibers: a weight-of-the-evidence approach. Regul. Toxicol. Pharmacol. 33, 319-342], well-conducted inhalation studies are very sensitive and rats may be more sensitive than humans in detecting the carcinogenic potential of MMVF. However, their arguments are highly questionable. The explanations of the IARC working group for preferring the newer inhalation studies are not sufficiently supported by the published data. Having in mind the higher sensitivity of humans compared to rats after inhalation of asbestos, more emphasis should have been given to the carcinogenic response after intraperitoneal injection.

Cancer risk assessment for oral exposure to PAH mixtures.

Cancer risk estimates for oral uptake of polycyclic aromatic hydrocarbons (PAHs) currently are based on risk estimates for benzo[a]pyrene (BAP). The potency of PAH mixtures often is calculated using relative potency values (BAP equivalency factors). We used recent oral carcinogenicity studies with BAP and coal tar mixtures, as well as older studies for a critical reappraisal of the current practice. A literature survey identified several carcinogenicity studies with oral and dermal exposure and lung implantation that allow a direct comparison of the carcinogenic potency of pure BAP and PAH mixtures. Moreover, when the PAH composition of the mixture has been analysed, prediction of the potency of PAH mixtures by BAP equivalency factors could be compared with the observed PAH potency. The analysis indicates that BAP equivalency factors do not describe adequately the potency of PAH mixtures and lead to underestimations of carcinogenic potency in most cases. Evaluation of several studies with various PAH mixtures revealed that the potency ratio between pure BAP and the PAH mixture in the same assay is highly dependent on the exposure pathway and the target organ, therefore potency estimates for PAH mixtures should be derived separately for oral, dermal and inhalative exposure using data from studies with the relevant pathway. A cancer slope factor for oral PAH exposure was derived based on data from a recent feeding study with coal tar mixtures. By using incidence data for all exposure-related tumours, a slope factor for humans of 11.5 (human excess risk per oral lifetime exposure with 1 mg BAP kg(-1)day(-1) in a PAH mixture) was obtained. Our analysis led to the conclusion that the contribution of BAP to the carcinogenic potency of the mixture depends on the exposure pathway and type of cancer observed but is relatively constant for various PAH mixtures from industrial sources. Thus, the derived oral slope factor is recommended to be used for the risk assessment of PAH-contaminated soils.

Tumor necrosis factor alpha sensitizes low epidermal growth factor receptor (EGFR)-expressing carcinomas for anti-EGFR therapy.

Analysis of 1,060 xenotransplants derived from cancer cell lines as wel as spontaneously occurring tumors from the larynx, pharynx, mammary gland, uterine cervix, and vulva revealed that tumor regression induced by treatment with monoclonal antibodies (EMD 55900 and EMD 72000 against the epidermal growth factor receptor (EGFR) could be enhanced by tumor necrosis factor alpha (TNF-alpha) treatment in vivo. Moreover, tumor that primarily do not respond to antibody treatment can be made suscep tible by additional TNF-alpha treatment. To investigate the in vivo effects of monoclonal antibodies, we treated tumors derived from cell lines (A431 and Detroit 562) as well as spontaneously occurring squamous cell carci nomas and adenocarcinomas (transplanted on NMRI-nu/nu mice) gener ally with EMD 55900 (40 microg/g mouse) and its humanized version EMD 72000 (40 microg/g mouse). When treated with EMD 55900 and EMD 72000 carcinomas with an EGFR concentration of > or = 70 fmol/mg protein showed significant reduction in tumor size compared with untreated controls. The degree of tumor regression correlated with the EGFR concentration of the tumor. In mice treated with TNF-alpha (0.5 microg/g mouse) and EMD 55900 72000 simultaneously, we observed enhanced antitumor effects up to complete tumor eradication. Carcinomas with an EGFR concentration <70 fmol/mg protein could be made susceptible to treatment with EMD 55900 and EMD 72000 by simultaneous treatment with TNF-alpha, resulting in a significant reduction in tumor size.

Iatrogenic left ventricular-right atrial fistula following mitral valve replacement and tricuspid annuloplasty: diagnosis by transthoracic and transesophageal echocardiography.

Acquired left ventricle-to-right atrium communications are a known complication of valvular heart surgery. Previous reports have described the clinical features and diagnosis using cardiac catheterization. We report two cases of acquired left ventricle-to-right atrium fistula following mitral valve replacement. Particular emphasis is placed on the diagnosis using transthoracic and transesophageal echocardiography, obviating the need for cardiac catheterization before repair.

mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats.

Human malignant mesotheliomas are induced almost exclusively by fibrous dusts. The nature of interactions between fibers and target cells, and the molecular mechanisms leading to tumorigenesis, are not yet understood. Here, the mRNA expression patterns at different stages of asbestos-induced carcinogenesis in rats were monitored by suppression subtractive hybridization (SSH) and array assay. Several genes were upregulated in pretumorous tissues from asbestos-treated rats, in asbestos-induced tumors and in cells treated with asbestos in vitro. The upregulation of the proto-oncogene c-myc, fra-1 and egfr in fiber-induced carcinogenesis was demonstrated at different stages of carcinogenesis. A possible role of Fra-1 as one of the dimeric proteins generating the AP-1 transcription factor was substantiated by its dose-dependent expression in mesothelial cells treated with asbestos in vitro. The upregulation of osteopontin (an extracellular matrix protein) and of zyxin and integrin-linked kinase (intracellular proteins associated with the focal adhesion contact), indicate that fibers may affect integrin-linked signal transduction and extracellular matrix proteins.

Peptidergic input to immunohistochemically-identified Renshaw cells.

Peptidergic influences on Renshaw cells were assessed in rat using gephyrin-immunoreactivity, as a Renshaw cell specific marker, in combination with substance P, calcitonin gene-related peptide- and nicotinic acetylcholine receptor-immunolabelling. An average of 3.9 substance P-, and 8.1 calcitonin gene-related peptide-, and 16.3 nicotinic acetylcholine receptor-immunoreactive close contacts or puncta were observed per Renshaw cell. Most appositions were somatic. These results provide neuroanatomical support for the peptidergic modulation of Renshaw cells.

DNA fingerprint analysis reveals differences in mutational patterns in experimentally induced rat peritoneal tumors, depending on the type of environmental mutagen.

We performed tumor DNA fingerprint analysis using the synthetic minisatellite probe S3315x2 based on the 33.15-repeat unit. The aim of the study was to investigate fingerprinting patterns of peritoneal tumors induced experimentally in Wistar rats by two carcinogens with unknown mechanism of action (crocidolite asbestos and nickel powder) and, as a positive control, benzo[a]pyrene. The carcinogens were administered intraperitoneally into rats. The banding patterns obtained with DNA from 71 peritoneal tumors were compared to the corresponding normal tissues. DNA derived from peritoneal tumors induced by the three carcinogens differed with respect to mutation frequencies and mutation patterns. The mutation frequencies in these tumors, revealed by DNA fingerprinting, were 18.2% for benzo[a]pyrene, 14.8% for crocidolite asbestos, and 40.9% for nickel powder. The alterations detected in the banding pattern of benzo[a]pyrene-induced peritoneal tumors were exclusively additional bands. On the contrary, in the DNA from asbestos-induced peritoneal tumors, only deletions of bands were observed on the autoradiographs. In the DNA from nickel-induced peritoneal tumors, both types of mutations occurred. The different mutation frequencies and mutation patterns appear to discriminate between benzo[a]pyrene, crocidolite asbestos, and nickel powder, and may be related to the mechanisms of action of these compounds.

Fiber-specific molecular features of tumors induced in rat peritoneum.

Molecular markers such as mutational spectra or mRNA expression patterns may give some indication of the mechanisms of carcinogenesis induced by fibers and other carcinogens. In our study, tumors were induced by application of crocidolite asbestos or benzo[a]pyrene (B[a]P) to rat peritoneum. DNA and RNA of these tumors were subjected to analysis of point mutations and to investigation of mRNA expression patterns. With both assays we found typical features depending on the type of carcinogen applied. The analysis of point mutations in the tumor suppressor gene p53 revealed mutations in the B[a]P-induced tumors. However, in the tumors induced by crocidolite asbestos that were of the same tumor type as those induced by B[a]P, mutations in p53 were not detectable. Every mutation detected on the DNA level causes an amino acid substitution within one of the functional domains of the tumor suppressor protein. Therefore, these mutations seem to be of biological relevance for tumor progression and indicate a difference in the carcinogenesis regarding the type of the carcinogenic substance. An additional specificity of crocidolite-induced tumors was detectable by analyzing the mRNA expression of the tumor suppressor gene WT1, which is known to be expressed in human mesothelial and mesothelioma cells. A relatively high amount of WT1 mRNA was measured by quantitative competitive reverse transcription-polymerase using RNA extracted from crocidolite-induced tumors. However, WT1 seems to be expressed on a rather low level in tumors induced by B[a]P.

Dose-response relationship of fibrous dusts in intraperitoneal studies.

The relationship between the number of fibers injected intraperitoneally and the occurrence of peritoneal mesotheliomas in rats was investigated using data from a series of carcinogenicity studies with several fibrous dusts. Based on observed tumor incidences ranging between 10 and 90%, the hypothesis of a common slope of dose-response relationships (parallel probit lines in probit analysis) cannot be rejected. In general, parallelism of probit lines is considered an indication of a common mode of action. Analysis of the shape of the dose-response relationship, with one apparent exception, shows virtually linear or superlinear behavior, i.e., from these data, there is no indication of a decrease in carcinogenic potency of an elementary carcinogenic unit at lower doses.

P53 mutations in tumours induced by intraperitoneal injection of crocidolite asbestos and benzo[a]pyrene in rats.

Mutation analysis of the tumour suppressor gene p53 in tumours induced in the peritoneal cavity of rats revealed differences in the mutational pattern with regard to the carcinogenic substances applied. In tumours induced by benzo[a]pyrene a considerable amount of p53 mutations resulting in an altered protein structure could be detected. For the development of these tumours an escape from the p53 mediated cell cycle control can be assumed. However, in tumours of the same tumour type induced by crocidolite asbestos no mutations could be observed. Since there were even no spontaneous p53 mutations detectable in this tumour group, it is obvious that in these tumours the escape from cell cycle control does not take place via inactivation of p53. Therefore, it is concluded that the molecular mechanisms of carcinogenesis and tumour development in this tumour type depend on the type of carcinogen applied.

[Current data and questions on the carcinogenicity of solid particles of diesel engine exhaust and other sources]. / Aktuelle Daten und Fragen zur Kanzerogenität von festen Partikeln aus Abgas von Dieselmotoren und anderen Quellen.

During the last few years, the findings could be confirmed which led to the conclusion that very fine solid particles which are not known to have an intrinsic toxicity, can induce lung tumours in rats. The carcinogenic potency seems to increase parallel to increasing specific surface area and decreasing particle size. However, many questions remain open. Organic compounds which contain many carcinogenic polycyclic aromatic hydrocarbons (PAH) can explain only less than 1% of the carcinogenic effect of diesel exhaust in the rat lung. Therefore, the carcinogenicity of diesel exhaust cannot be reduced by an oxidizing catalyst. It burns a part of the organic substances adsorbed on the surface of the carbonaceous core of diesel particles and thus helps to follow the low standard for particles in diesel emissions, but without reduction of the carcinogenic potential because it does not reduce the emission of the insoluble carcinogenic part of the particles, the elementary carbonaceous core. The search for a hypothetical threshold for the carcinogenicity of particles in the rat lung aims at the determination of mechanisms which are preconditions for the development of a tumour. If certain doses do not induce such lesions they are considered safe. Chronic inflammation, proliferation of epithelial cells and fibrosis are discussed to be preconditions. However, the pathways which lead to inflammation and proliferation could proceed independent of the molecular carcinogenesis. Therefore, a cancer risk from diesel particles and other very fine particles may occur under environmental conditions. If the positive epidemiologic studies are relevant, a lung cancer risk for humans can be calculated which is higher than that calculated from inhalation studies with rats.

Effect of subchronic 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on immune system and target gene responses in mice: calculation of benchmark doses for CYP1A1 and CYP1A2 related enzyme activities.

The dose-effect relationships were analysed for several noncarcinogenic endpoints, such as immunological and biochemical responses at subchronic, low dose exposure of female C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The animals were treated i.p. with TCDD according to the initial- and maintenance-dose principal for a period of 135 days. The initial doses were 1, 10 and 100 ng TCDD/kg, the weekly maintenance doses were 0.2, 2 and 20 ng TCDD/kg, respectively. At days 23, 79 and 135 of TCDD/kg, treatment 10 animals of each dose group were killed. As immunological parameters the number of thymocytes and the pattern of thymocyte subpopulations were determined. In liver, lung and thymus, mRNA expression of TGF-alpha, TGF-beta(1), TGF-beta(2), TGF-beta(3), TNF-alpha, IL-1 beta and different CYP1 isoforms (CYP1A1, CYP1A2, CYP1B1) was analysed. In the livers, activities of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) were measured. TCDD content in the liver was determined. The main results are summarized as follows: (1) The TCDD doses were not sufficient to elicit dose-dependent changes of pattern of thymocyte subpopulation. (2) TCDD failed to change the mRNA expression of TGF-alpha, TGF-beta and TNF-alpha, but led to an increase of IL-1 beta mRNA expression in liver, lung and thymus. The results show that the TCDD induced IL-1 beta mRNA increase is at least as sensitive a marker as the induction of CYP1A isoforms. (3) The expression of CYP1B1 mRNA remained unchanged at the doses tested, while CYP1A1 and CYP1A2 mRNA expression was dose-dependently enhanced. EROD and MROD activities in the liver paralleled the increases of CYP1A1 and CYP1A2 mRNA expression. (4) Regression analysis of the data showed that most of the parameters tested fit a linear model. (5) From the data, a benchmark dose for EROD/MROD activities in the livers of female C57BL/6 mice of about 0.03 ng TCDD/kg per day was calculated.

Results of current intraperitoneal carcinogenicity studies with mineral and vitreous fibres.

The study includes some 50 groups of male or female Wistar rats tested in three series. Except for one untreated group and 3 vehicle control groups, the animals were injected intraperitoneally (i.p.) once or repeatedly with dust suspensions and then examined, after lifetime observation up to 30 months, for tumours in the abdominal cavity. 1 granular dust (silicon carbide), 2 asbestos dusts (crocidolite, tremolite) and 11 vitreous fibre dust samples were administered. 5 of the vitreous fibre types were fine fibre fractions from 4 commercial insulation wools and 1 experimental wool, the others were prepared by milling glass microfibres, which have, per se, a small diameter range. The dosage per rat differed over a wide range in accordance with experience from earlier studies. The lowest dose was 0.04 x 10(9) crocidolite fibres in 0.5 mg dust, and the highest amounted to 20 x 10(9) glass fibres in 1000 mg divided into 40 weekly injections. Two mesotheliomas were found in a total of 395 rats treated with saline or granular silicon carbide (250 or 1000 mg). Eleven fibre dusts produced dose-dependent mesotheliomas at rates of up to 97 %, but the calculated fibre number > 5 micrometers in length required for inducing a 25 % tumour risk differed between the fibre samples tested in the relation of 1 to about 1000. UICC-like crocidolite heads the ranking order; the glass fibre B-01, which possesses a low durability in the body, ends it together with a rather thin sample of glass fibre type B-09. The stone fibre MMVF-21 takes a high place in the ranking order, similar to the tremolite sample. The results correspond to those of earlier i.p. tests.

Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion.

The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.