Systemic RNAi in Caenorhabditis elegans.

RNA interference (RNAi) in Caenorhabditis elegans induced by ingestion or injection of double-stranded RNA (dsRNA) spreads throughout the organism and is even transmitted to the progeny. We have identified two proteins required for spreading of RNAi, SID-1 and SID-2, whose structure, subcellular localization, and expression pattern have been informative for how dsRNA can be transported into and between cells. SID-1 is a transmembrane protein that functions as a pore or channel that transports dsRNA into and out of cells. Proteins homologous to SID-1 are present in a wide range of invertebrate and vertebrate animals but are absent from plants. SID-2 is a small transmembrane protein that is expressed in the gut and localizes strongly to the luminal membrane where it appears to act as a receptor for uptake of dsRNA from the environment. Characterization of SID-2 activity in a variety of Caenorhabditis nematodes indicates that C. elegans SID-2 may have a novel activity.

CDC-42 regulates PAR protein localization and function to control cellular and embryonic polarity in C. elegans.

BACKGROUND: The polarization of the anterior-posterior axis (A-P) of the Caenorhabditis elegans zygote depends on the activity of the par genes and the presence of intact microfilaments. Functional links between the PAR proteins and the cytoskeleton, however, have not been fully explored. It has recently been shown that in mammalian cells, some PAR homologs form a complex with activated Cdc42, a Rho GTPase that is implicated in the control of actin organization and cellular polarity. A role for Cdc42 in the establishment of embryonic polarity in C. elegans has not been described. RESULTS: To investigate the function of Cdc42 in the control of cellular and embryonic polarity in C. elegans, we used RNA-mediated interference (RNAi) to inhibit cdc-42 activity in the early embryo. Here, we demonstrate that RNAi of cdc-42 disrupts manifestations of polarity in the early embryo, that these phenotypes depend on par-2 and par-3 gene function, and that cdc-42 is required for the localization of the PAR proteins. CONCLUSIONS: Our genetic analysis of the regulatory relationships between cdc-42 and the par genes demonstrates that Cdc42 organizes embryonic polarity by controlling the localization and activity of the PAR proteins. Combined with the recent biochemical analysis of their mammalian homologs, these results simultaneously identify both a regulator of the PAR proteins, activated Cdc42, and effectors for Cdc42, the PAR complex.

Quantitative analysis of mRNA amplification by in vitro transcription.

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Evaluation of normalization procedures for oligonucleotide array data based on spiked cRNA controls.

BACKGROUND: Affymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization. RESULTS: We first explore the conditions for validity of the 'constant mean assumption', the key assumption underlying current normalization methods. We introduce 'frequency normalization', a 'spike-in'-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define 'scaled frequency', a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages. CONCLUSIONS: Scaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.

Genomic analysis of gene expression in C. elegans.

Until now, genome-wide transcriptional profiling has been limited to single-cell organisms. The nematode Caenorhabditis elegans is a well-characterized metazoan in which the expression of all genes can be monitored by oligonucleotide arrays. We used such arrays to quantitate the expression of C. elegans genes throughout the development of this organism. The results provide an estimate of the number of expressed genes in the nematode, reveal relations between gene function and gene expression that can guide analysis of uncharacterized worm genes, and demonstrate a shift in expression from evolutionarily conserved genes to worm-specific genes over the course of development.

Gene silencing: shrinking the black box of RNAi.

The mysterious mechanism whereby the mere presence of double-stranded RNA can inactivate specific genes is yielding its secrets. Recent results identifying cellular components required for RNAi in Caenorhabditis elegans indicate that the mechanism is conserved, ancient and may provide a defense against selfish DNA.

Epidemiology, stage at diagnosis, and tumor biology of breast carcinoma in multiracial and multiethnic populations.

All women, regardless of their racial or ethnic origin or heritage, are at risk of developing breast cancer. Variations in breast carcinoma incidence rates among multicultural populations suggest that etiologic factors differ in their biologic expression and impact on disease outcome. Key among those factors that affect breast carcinoma development are the roles of genetics and the environment, the reproductive experience and the effects of endogenous and exogenous hormones in women, the change in immune status and host vulnerability, and the biologic determinants of breast carcinoma. Cultural dynamics, sociodemographic differences, and behavioral characteristics across population subgroups modulate how biologic disease is expressed among different races and ethnic groups. These interactions contribute to the observed variations in breast carcinoma incidence, mortality, and survival. Stage, a measure of disease status, is used to assess prognosis, plan treatment, and evaluate outcome. Numerous studies have reported a more advanced stage of breast carcinoma at diagnosis in racial/ethnic subgroups, especially among women from African American, Hispanic, American Indian, and native Hawaiian cultures. Factors associated with advanced stage at diagnosis in multicultural populations range from changes in the basic biological characteristics at the molecular and cellular level, to more complex behavioral attributes unique to a particular multicultural population, to societal issues-such as access to care and socioeconomic conditions-all of which impact on the health measure called "stage at diagnosis." Rapid advancements in knowledge of cancer biology and of genetic markers and tumor products are providing new mechanisms for identifying etiologic pathways that can be utilized for better screening, detection, treatment and monitoring of disease. Further studies are needed that evaluate the biologic and molecular alterations in tumor development, progression, and response to therapy. Public health attention needs to be directed toward the societal influences that impact breast carcinoma development, as well as augmenting recognition of the need for culturally appropriate, broad-based behavioral changes at the community level. In addition, continued efforts are needed to ensure the inclusion of multicultural population subgroups and minority investigators in all aspects of research-basic, clinical and applied.

Genetics: a touch of elegance with RNAi.

RNA-mediated gene interference (RNAi), a rapid, convenient tool for inhibiting gene function in Caenorhabditis elegans, has recently been shown to work in other organisms.

Hox gene expression in a single Caenorhabditis elegans cell is regulated by a caudal homolog and intercellular signals that inhibit wnt signaling.

In Caenorhabditis elegans males, a row of epidermal precursor cells called seam cells generates a pattern of cuticular alae in anterior body regions and neural sensilla called rays in the posterior. The Hox gene mab-5 is required for two posterior seam cells, V5 and V6, to generate rays. In mab-5 mutant males, V5 and V6 do not generate sensory ray lineages but instead generate lineages that lead to alae. Here we show that two independent regulatory pathways can activate mab-5 expression in the V cells. First, the caudal homolog pal-1 turns on mab-5 in V6 during embryogenesis. Second, a Wnt signaling pathway is capable of activating mab-5 in the V cells during postembryonic development; however, during normal development Wnt signaling is inhibited by signals from neighboring V cells. The inhibition of this Wnt signaling pathway by lateral signals between the V cells limits the number of rays in the animal and also determines the position of the boundary between alae and rays.

The maternal par genes and the segregation of cell fate specification activities in early Caenorhabditis elegans embryos.

After fertilization in C. elegans, activities encoded by the maternally expressed par genes appear to establish cellular and embryonic polarity. Loss-of-function mutations in the par genes disrupt anterior-posterior (a-p) asymmetries in early embryos and result in highly abnormal patterns of cell fate. Little is known about how the early asymmetry defects are related to the cell fate patterning defects in par mutant embryos, or about how the par gene products affect the localization and activities of developmental regulators known to specify the cell fate patterns made by individual blastomeres. Examples of such regulators of blastomere identity include the maternal proteins MEX-3 and GLP-1, expressed at high levels anteriorly, and SKN-1 and PAL-1, expressed at high levels posteriorly in early embryos. To better define par gene functions, we examined the expression patterns of MEX-3, PAL-1 and SKN-1, and we analyzed mex-3, pal-1, skn-1 and glp-1 activities in par mutant embryos. We have found that mutational inactivation of each par gene results in a unique phenotype, but in no case do we observe a complete loss of a-p asymmetry. We conclude that no one par gene is required for all a-p asymmetry and we suggest that, in some cases, the par genes act independently of each other to control cell fate patterning and polarity. Finally, we discuss the implications of our findings for understanding how the initial establishment of polarity in the zygote by the par gene products leads to the proper localization of more specifically acting regulators of blastomere identity.

Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos.

The early asymmetric cleavages of Caenorhabditis elegans embryos produce blastomeres with distinct developmental potentials. Here, we show that the caudal-like homeodomain protein PAL-1 is required to specify the somatic identity of one posterior blastomere in the 4 cell embryo. We find that pal-1 activity is sequentially restricted to this blastomere. First, at the 4 cell stage, it is translated only in the two posterior blastomeres. Then, its function is restricted to one of these blastomeres. This second targeting step is dependent on the activities of the posteriorly localized SKN-1 and asymmetrically segregated PIE-1 proteins. We propose that the segregation of PIE-1, combined with the temporal decay of SKN-1, targets pal-1 activity to this posterior lineage, thus coupling the regulation of this conserved posterior patterning gene to asymmetric cell cleavages.

Determinants of black/white differences in colon cancer survival.

BACKGROUND: Blacks have lower survival rates for colon cancer than whites, possibly related to more advanced stages of disease at diagnosis and to socioeconomic differences between blacks and whites. While the black/white difference in colon cancer survival is well documented, the few studies that have investigated this difference have been limited by the modest number and type of explanatory factors that were considered. PURPOSE: We analyzed data from the National Cancer Institute Black/White Cancer Survival Study to determine 1) what characteristics might contribute to the racial difference in colon cancer survival and 2) if a survival disparity remained between black and white patients after adjustment was made for these characteristics. METHODS: This prospective study included 454 blacks and a stratified random sample of 521 whites, aged 20-79 years, with cancer of the colon diagnosed from January 1, 1985, through December 31, 1986, and who were residents of the metropolitan areas of Atlanta, New Orleans, and San Francisco/Oakland. Follow-up was truncated on December 31, 1990. Cox proportional hazards regression was used to estimate the death rate among blacks relative to that among whites after adjustment for potential explanatory factors, including sociodemographic factors, concurrent (comorbid) medical conditions, stage at diagnosis, tumor characteristics, and treatment. All P values were calculated from two-tailed tests of statistical significance. RESULTS: After adjustment for age, sex, and geographic area, the black-to-white mortality hazard ratio (HR) was 1.5 (95% confidence interval [CI] = 1.2-1.9), indicating that the risk of death among black patients was 50% higher than that among white patients. Further adjustment for stage reduced the excess cancer mortality to 20% (HR = 1.2; 95% CI = 1.0-1.5), decreasing the overall racial difference in excess mortality from 50% to 20% or to a 60% reduction in excess mortality. Although adjustment for poverty reduced the excess mortality by 20%, adjusting for both stage and poverty did not further reduce the racial difference. Among patients with stages II and III disease, blacks had lower survival rates than whites (HR = 1.8; 95% CI = 1.0-3.1 and HR - 1.5; 95% CI = 1.0-2.3, respectively). Among those patients with metastatic disease (stage IV), survival was similar for whites and blacks. CONCLUSIONS: Stage at diagnosis accounted for more than half of the excess colon cancer mortality observed among blacks. Poverty and other socioeconomic conditions, general health status, tumor characteristics, and general patterns of treatment did not further explain the remaining survival disadvantage among blacks. IMPLICATIONS: Because the racial disparity was confined to earlier stages, future studies should investigate whether blacks have more advanced disease at diagnosis and whether less aggressive treatment is provided because of understanding.

Specification of anteroposterior cell fates in Caenorhabditis elegans by Drosophila Hox proteins.

Antennapedia class homeobox (Hox) genes specify cell fates in successive anteroposterior body domains in vertebrates, insects and nematodes. The DNA-binding homeodomain sequences are very similar between vertebrate and Drosophila Hox proteins, and this similarity allows vertebrate Hox proteins to function in Drosophila. In contrast, the Caenorhabditis elegans homeodomains are substantially divergent. Further, C. elegans differs from both insects and vertebrates in having a non-segmented body as well as a distinctive mode of development that involves asymmetric early cleavages and invariant cell lineages. Here we report that, despite these differences, Drosophila Hox proteins expressed in C. elegans can substitute for C. elegans Hox proteins in the control of three different cell-fate decisions: the regulation of cell migration, the specification of serotonergic neurons, and the specification of a sensory structure. We also show that the specificity of one C. elegans Hox protein is partly determined by two amino acids that have been implicated in sequence-specific DNA binding. Together these findings suggest that factors important for target recognition by specific Hox proteins have been conserved throughout much of the animal kingdom.

Anatomic site distribution of colon cancer by race and other colon cancer risk factors.

PURPOSE: Black patients with colon cancer are more likely to have poorer survival from colon cancer than are white patients. To determine whether anatomic site differences might contribute to survival differences, we compared anatomic site distributions of black and white patients. METHODS: As part of the Black/White Cancer Survival Study, we collected medical record data for 1,045 patients from Atlanta, New Orleans, and San Francisco/Oakland, newly diagnosed in 1985 or 1986 and interviewed 745 of them. RESULTS: In polychotomous logistic regression analysis, site was related to stage, grade, and histologic type and among women with age, parity, and possibly smoking. However, it was not related to race, except perhaps among men age 65 and older, among whom blacks were somewhat likely to have more transverse and distal, not proximal, cancer. These relations were consistent across subgroups and were independent of other factors examined. CONCLUSION: Results suggest that site differences are unlikely to contribute to poorer survival commonly observed among black colon cancer patients in the United States.

Histological characteristics of breast carcinoma in blacks and whites.

Tumor characteristics of 963 newly diagnosed invasive breast cancer cases from the population-based Black/White Cancer Survival Study were evaluated. Representative slides of the tumors were requested from all participating hospitals of three metropolitan areas and reviewed by one expert pathologist, blinded in regard to the age and race of patients. Nine tumor characteristics were evaluated for black and white patients. After adjusting for age, stage, and metropolitan area, blacks were significantly more likely to have high grade nuclear atypia [odds ratio (OR) = 1.97, 95% confidence interval (CI) = 1.27-3.04]; high mitotic activity (OR = 2.05, 95% CI = 1.34-3.14), grade 3 tumors (OR = 1.58, 95% CI = 1.02-2.45), and more necrosis (OR = 1.51, 95% CI = 1.16-1.98); and less likely to have well defined tubular formation (OR = 0.57, 95% CI = 0.42-0.77), marked fibrosis (OR = 0.65, 95% CI = 0.45-0.94), and positive estrogen receptor status (OR = 0.78, 95% CI = 0.58-1.05). These black/white differences remained after controlling for socioeconomic status (SES), body mass index, use of alcohol and tobacco, reproductive experience, and health care access and utilization. No significant racial differences were found for blood vessel invasion and lymphatic invasion. Although white women of high SES had more favorable tumors than those of low SES, the same pattern was not observed for blacks. High SES black women had statistically nonsignificant elevated ORs of a high mitotic index and tumor grade. These racial differences in tumor biology may have etiological and clinical implications.

Breast cancer: factors associated with stage at diagnosis in black and white women. Black/White Cancer Survival Study Group.

BACKGROUND: Numerous studies have reported differences in cancer staging at diagnosis and in survival between Black and White patients with breast cancer. Utilizing data obtained from the National Cancer Institute's (NCI's) Black/White Cancer Survival Study for the period 1985-1986, a new study is presented here that systematically examines multiple explanatory factors (e.g., lack of mammograms) associated with these cancer-staging differences. PURPOSE: We evaluated within a single study the relationship of selected demographic, lifestyle, antecedent medical experiences, and health care access factors to cancer staging at diagnosis in Black and White breast cancer patients. METHODS: Data utilized in this population-based cohort study of 1222 eligible women (649 Black and 573 White) newly diagnosed for the period 1985-1986 with histologically confirmed primary breast cancer were obtained from the NCI's Black/White Cancer Survival Study. Sources of data included abstracts of hospital medical records, central review of histology slides by a study consultant pathologist, and patient interviews obtained from three metropolitan areas: Atlanta, New Orleans, and San Francisco-Oakland. Within each area, 70% of all Black incident cases were randomly selected, and a sample of White cases, frequency matched by age groups (20-49 years, 50-64 years, and 65-79 years), was selected for comparison. Stage of breast cancer at diagnosis was classified according to the international tumor-lymph node-metastases (TNM) system. Statistical models utilized in this study included the log-linear and polychotomous logistic regression with multiple predictor variables. RESULTS: Factors associated with cancer staging were differentially expressed in Blacks and Whites. Indicators of access to health care, a lack of mammograms, and an increased body mass index significantly (P < .02) contributed to stage differences in Blacks, whereas income was marginally associated (P = .06) with stage for Whites only. Nuclear grade, having a breast examination by a physician, and a history of patient delay explained approximately 50% of the excess risk for stage III-IV cancer versus stage I-IIN0 cancer among Blacks compared with Whites (odds ratio reduction from 2.19 to 1.68). CONCLUSION: These findings suggest that no single factor or group of factors can explain more than half of the race-stage differences noted in this study with respect to Black and White breast cancer patients.

Treatment plans for black and white women with stage II node-positive breast cancer. The National Cancer Institute Black/White Cancer Survival Study experience.

BACKGROUND: The National Cancer Institute Black/White Cancer Survival Study began patient accrual in 1985 and was designed to investigate factors that might contribute to the observed racial differences in survival for cancer of the breast, uterine corpus, colon, and bladder. METHODS: To determine whether there were racial differences in treatment in a clinically homogeneous set of patients, 305 (25%) of the 1222 women in this study with Stage II node-positive (N+) breast cancer were evaluated. RESULTS: Patient characteristics for blacks and whites were similar for age, metropolitan area of residence, tumor size, extent of nodal involvement, and steroid receptors. Differences in histologic findings, tumor grade, and nuclear atypia were observed. Blacks had a higher frequency of comorbid conditions, especially hypertension (P < 0.00001). Fewer blacks underwent breast-conserving surgery (P = 0.004). In a multivariate analysis, race was no longer a significant factor in the selection of primary treatment, but education and metropolitan area of residence remained significant. Blacks and whites received similar postoperative systemic therapy, with combination chemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil) and tamoxifen, the most common cytotoxic and endocrine therapies used. CONCLUSIONS: The National Cancer Institute consensus statement concerning adjuvant therapy for breast cancer was published in the middle of the 2-year period that study cases were accrued, and treatment plans in this study generally agreed with consensus guidelines. Should survival differences in black and white patients with Stage II N+ disease in this study be found, they are unlikely to be attributable to differences in initial or postoperative treatment.

Differences between black and white women with breast cancer in time from symptom recognition to medical consultation. Black/White Cancer Survival Study Group.

BACKGROUND: Studies in the United States have reported that Black women have higher fatality rates than White women following a diagnosis of breast cancer and are more likely to be diagnosed with late-stage cancers. PURPOSE: To evaluate reasons for these racial differences, we explored the difference between Black and White women in the length of time from symptom recognition to initial medical consultation. We also evaluated the extent to which other factors related to the length of this interval might contribute to any observed racial difference. METHODS: As part of a collaborative study of differences in the survival rates of Black patients and White patients with cancer, we interviewed a sample of 410 Black women and 325 White women from Atlanta, New Orleans, and San Francisco/Oakland who were newly diagnosed in 1985 or 1986 with invasive breast cancer. Retrospective data were collected on symptoms, dates of symptom recognition and initial medical consultation, and several other factors which may affect the interval between symptom recognition and medical consultation. Data were analyzed as if from a follow-up study, using product limit procedures and proportional hazards regression. RESULTS: At diagnosis, Black women with breast cancer were two times more likely to have stage IV breast cancer and one and one-half times more likely to have stage III breast cancer than White women with breast cancer and were only approximately one-half as likely to have stage I breast cancer. Similarly, Black women were almost twice as likely as White women to have tumors that were larger than 5 cm or tumors that had extensions to the chest wall or skin at presentation. However, the average rate at which Black women with breast cancer obtained an initial medical consultation lagged behind that for White women by only a slight but statistically significant difference (15%). The median time between symptom recognition and medical consultation was slightly longer for Black women (16 days) than for White women (14 days) (P = .06). Adjustment for other characteristics predictive of the length of this interval had little effect on racial differences. The racial differences tended to vary somewhat by age and metropolitan area, suggesting that the results may not apply equally to all demographic subgroups and regions in the United States. CONCLUSION: This small difference in the time from symptom recognition to medical consultation is unlikely to account for the large racial differences in survival rates and in stage of disease at the time of diagnosis.