From symmetry to asymmetry: Phylogenetic patterns of asymmetry variation in animals and their evolutionary significance

Phylogenetic analyses of asymmetry variation offer a powerful tool for exploring the interplay between ontogeny and evolution because (i) conspicuous asymmetries exist in many higher metazoans with widely varying modes of development, (ii) patterns of bilateral variation within species may identify genetically and environmentally triggered asymmetries, and (iii) asymmetries arising at different times during development may be more sensitive to internal cytoplasmic inhomogeneities compared to external environmental stimuli. Using four broadly comparable asymmetry states (symmetry, antisymmetry, dextral, and sinistral), and two stages at which asymmetry appears developmentally (larval and postlarval), I evaluated relations between ontogenetic and phylogenetic patterns of asymmetry variation. Among 140 inferred phylogenetic transitions between asymmetry states, recorded from 11 classes in five phyla, directional asymmetry (dextral or sinistral) evolved directly from symmetrical ancestors proportionally more frequently among larval asymmetries. In contrast, antisymmetry, either as an end state or as a transitional stage preceding directional asymmetry, was confined primarily to postlarval asymmetries. The ontogenetic origin of asymmetry thus significantly influences its subsequent evolution. Furthermore, because antisymmetry typically signals an environmentally triggered asymmetry, the phylogenetic transition from antisymmetry to directional asymmetry suggests that many cases of laterally fixed asymmetries evolved via genetic assimilation.

Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.

The activation of plant defensive genes in leaves of tomato plants in response to herbivore damage or mechanical wounding is mediated by a mobile 18-amino acid polypeptide signal called systemin. Systemin is derived from a larger, 200-amino acid precursor called prosystemin, similar to polypeptide hormones and soluble growth factors in animals. Systemin activates a lipid-based signaling cascade, also analogous to signaling systems found in animals. In plants, linolenic acid is released from membranes and is converted to the oxylipins phytodienoic acid and jasmonic acid through the octadecanoid pathway. Plant oxylipins are structural analogs of animal prostaglandins which are derived from arachidonic acid in response to various signals, including polypeptide factors. Constitutive overexpression of the prosystemin gene in transgenic tomato plants resulted in the overproduction of prosystemin and the abnormal release of systemin, conferring a constitutive overproduction of several systemic wound-response proteins (SWRPs). The data indicate that systemin is a master signal for defense against attacking herbivores. The same defensive proteins induced by wounding are synthesized in response to oligosaccharide elicitors that are generated in leaf cells in response to pathogen attacks. Inhibitors of the octadecanoid pathway, and a mutation that interrupts this pathway, block the induction of SWRPs by wounding, systemin, and oligosaccharide elicitors, indicating that the octadecanoid pathway is essential for the activation of defense genes by all of these signals. The tomato mutant line that is functionally deficient in the octadecanoid pathway is highly susceptible to attacks by Manduca sexta larvae. The similarities between the defense signaling pathway in tomato leaves and those of the defense signaling pathways of macrophages and mast cells of animals suggests that both the plant and animal pathways may have evolved from a common ancestral origin.

Prostate cancer in a transgenic mouse.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.

Fine mapping of colon tumor susceptibility (Scc) genes in the mouse, different from the genes known to be somatically mutated in colon cancer.

The predisposition to colon cancer is multigenetically controlled in animals and probably also in humans. We have analyzed the multigenic control of susceptibility to 1,2-dimethylhydrazine-induced colon tumors in mice by using a set of 20 homozygous CcS/Dem recombinant congenic strains, each of which contains a different random subset of approximately 12.5% of genes from the susceptible strain STS/A and 87.5% of genes from the relatively resistant strain BALB/cHeA. Some CcS/Dem strains received the alleles from the susceptible strain STS/A at one or more of the multiple colon tumor susceptibility loci and are susceptible, whereas others are resistant. Linkage analysis shows that these susceptibility genes are different from the mouse homologs of the genes known to be somatically mutated in human colon cancer (KRAS2, TP53, DCC, MCC, APC, MSH2, and probably also MLH1). Different subsets of genes control tumor numbers and size. Two colon cancer susceptibility genes, Scc1 and Scc2, map to mouse chromosome 2. The Scc1 locus has been mapped to a narrow region of 2.4 centimorgans (90% confidence interval).

A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers

BRCA1 and BRCA2 carriers are at increased risk for both breast and ovarian cancer, but estimates of lifetime risk vary widely, suggesting their penetrance is modified by other genetic and/or environmental factors. The BRCA1 and BRCA2 proteins function in DNA repair in conjunction with RAD51. A preliminary report suggested that a single nucleotide polymorphism in the 5′ untranslated region of RAD51 (135C/G) increases breast cancer risk in BRCA1 and BRCA2 carriers. To investigate this effect we studied 257 female Ashkenazi Jewish carriers of one of the common BRCA1 (185delAG, 5382insC) or BRCA2 (6174delT) mutations. Of this group, 164 were affected with breast and/or ovarian cancer and 93 were unaffected. RAD51 genotyping was performed on all subjects. Among BRCA1 carriers, RAD51-135C frequency was similar in healthy and affected women [6.1% (3 of 49) and 9.9% (12 of 121), respectively], and RAD-135C did not influence age of cancer diagnosis [Hazard ratio (HR) = 1.18 for disease in RAD51-135C heterozygotes, not significant]. However, in BRCA2 carriers, RAD51-135C heterozygote frequency in affected women was 17.4% (8 of 46) compared with 4.9% (2 of 41) in unaffected women (P = 0.07). Survival analysis in BRCA2 carriers showed RAD51-135C increased risk of breast and/or ovarian cancer with an HR of 4.0 [95% confidence interval 1.6–9.8, P = 0.003]. This effect was largely due to increased breast cancer risk with an HR of 3.46 (95% confidence interval 1.3–9.2, P = 0.01) for breast cancer in BRCA2 carriers who were RAD51-135C heterozygotes. RAD51 status did not affect ovarian cancer risk. These results show RAD51-135C is a clinically significant modifier of BRCA2 penetrance, specifically in raising breast cancer risk at younger ages.

Low frequency of E-cadherin alterations in familial breast cancer

In order to explore the possible role of E-cadherin in familial cancer, 19 familial breast cancer patients, whose tumours demonstrated loss of heterozygosity (LOH) at the E-cadherin locus, were screened for germline mutations. No pathogenic germline alterations were detected in these individuals. However, a somatic mutation was found (49-2A→C) in one of the tumours. This tumour showed a pattern of both ductal and lobular histology. Another 10 families with cases of breast, gastric and colon cancer were also screened for germline mutations, and no mutations were found. A missense mutation in exon 12 of E-cadherin (1774G→A; Ala592Thr) was previously found in one family with diffuse gastric cancer, and colon and breast cancer. An allelic association study was performed to determine whether the Ala592Thr alteration predisposes to breast cancer. In total, we studied 484 familial breast cancer patients, 614 sporadic breast cancer patients and 497 control individuals. The frequencies of this alteration were similar in these groups. However, a correlation between the Ala592Thr alteration and ductal comedo-type tumour was seen. These results, together with previously reported studies, indicate that germline mutations and, more commonly, somatic mutations in E-cadherin may have an influence on the behaviour of the tumours, rather than predispose to breast cancer.

Signature p53 mutation at DNA cross-linking sites in 8-methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancers.

A combination of psoralen and ultraviolet A radiation (PUVA) is widely used in the treatment of psoriasis. However, PUVA treatment increases the risk of developing skin cancer in psoriasis patients and induces skin cancer in mice. Since the DNA damage induced by PUVA is quite different from that induced by UV, we investigated whether PUVA-induced mouse skin cancers display carcinogen-specific mutations in the p53 tumor suppressor gene. The results indicated that 10 of 13 (77%) PUVA-induced skin tumors contained missense mutations predominantly at exons 6 and 7. In contrast, tumor-adjacent, PUVA-exposed skin from tumor-bearing animals did not exhibit p53 mutation in exons 4-8. Interestingly, about 40% of all mutations in PUVA-induced skin tumors occurred at 5'-TA sites, and an equal number of mutations occurred at one base flanking 5'TA or 5'-TAT sites. Since PUVA induces DNA cross-links exclusively at these sites and since UV "signature" mutations were rarely detected in PUVA-induced skin cancers, we can conclude that PUVA acts as a carcinogen by inducing unique PUVA signature mutations in p53. This finding may have implications for identifying the etiology of skin cancer in psoriasis patients who have undergone PUVA therapy.

Cellular aging, destabilization, and cancer.

Three major characteristics of aging in animals are a slowdown of cell proliferation, an increase in residual bodies associated with age pigments, and a marked increase in the likelihood of neoplastic transformation. The 28 L subline of the NIH 3T3 line of mouse embryo fibroblasts exhibits all these characteristics when held at confluence for extended periods. The impairment of proliferation is the first behavioral characteristic detected in low density subcultures from the confluent cultures, and it persists through many cell generations of exponential multiplication. There is an equal degree of growth impairment among replicate cultures (lineages) recovered after each of 2 successive rounds of confluence, although heterogeneity appears after the third round. The growth impairment pervades the entire cell population of each lineage. The degree and duration of impairment increase with repeated rounds of confluence. A marked increase of residual bodies characteristic of age pigments occurs in the cytoplasm of all the cells kept under prolonged confluence. Neoplastic transformation first appears as foci of multilayered cells on a monolayered background of nontransformed cells. The transformed cells arise at different times in the lineages and originate from a very small fraction of the population. The transformed cells selectively overgrow the entire population in successive rounds of confluence leading to an increase in saturation density of each lineage at different times. Under cloning conditions, isolated colonies of transformed cells develop more slowly than colonies of nontransformed cells but eventually reach a higher population density. The regularity of persistent growth impairment among the lineages and the appearance of large numbers of residual bodies in all the cells of each population are more characteristic of an epigenetic process than of specific local mutations. although random chromosomal lesions cannot be ruled out. By contrast, the low frequency and stochastic character of neoplastic transformation are consistent with a conventional genetic origin. The advent in long-term confluent NIH 3T3 cultures of three cardinal characteristics of cellular aging in vivo recommends it as a model for aging cells.

Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.