Mélina Arguin

β1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms

The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of β1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-XL, Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak–Src association, and a sustained/enhanced activation of p38β, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-XL and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38β activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38β activation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by β1 integrins/Fak, and is crucial for survival; and (6) β1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, β1 integrin/Fak/Src signaling translates into integrated mediating functions of p38β activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.

Intestinal Epithelial Cancer Cell Anoikis Resistance: EGFR-Mediated Sustained Activation of Src Overrides Fak-Dependent Signaling to MEK/Erk and/or PI3-K/Akt-1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009.

Male-driven de novo mutations in haploid germ cells

At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA end-joining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.

Genome-wide mapping of DNA strand breaks.

Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed “damaged DNA immunoprecipitation” (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

Instability of Trinucleotidic Repeats During Chromatin Remodeling in Spermatids

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.

Breaking news from spermatids.

During the haploid phase of spermatogenesis, spermatids undergo a complex remodeling of the paternal genome involving the finely orchestrated replacement of histones by the highly-basic protamines. The associated striking change in DNA topology is characterized by a transient surge of both single- and double-stranded DNA breaks in the whole population of spermatids which are repaired before spermiation. These transient DNA breaks are now considered part of the normal differentiation program of these cells. Despite an increasing interest in the study of spermiogenesis in the last decade and the potential threat to the haploid genome, the origin of these DNA breaks still remains elusive. This review briefly outlines the current hypotheses regarding possible mechanisms that may lead to such transient DNA fragmentation including torsional stress, enzyme-induced breaks, apoptosis-like processes or oxidative stress. A better understanding of the origin of these DNA breaks will lead to further investigations on the genetic instability and mutagenic potential induced by the chromatin remodeling.RésuméLors de la phase haploïde de la spermatogenèse, les spermatides subissent un remodelage complexe du génome paternel impliquant un remplacement finement orchestré des histones par des protamines hautement basiques. Le changement topologique de l’ADN associé est caractérisé par une augmentation transitoire de cassures simple et double brins de l’ADN dans l’entière population des spermatides qui sont réparées avant la spermiation. Ces cassures transitoires de l’ADN sont maintenant considérées comme faisant partie du processus normal de différenciation de ces cellules. Malgré un intérêt croissant dans l’étude de la spermiogenèse ces 10 dernières années et la menace potentielle pour le génome haploïde, l’origine de ces cassures d’ADN reste encore incertaine. Cette revue décrit brièvement les hypothèses actuelles concernant les mécanismes possibles qui pourraient mener à cette fragmentation transitoire de l’ADN incluant le stress torsionnel, les cassures enzymatiques, des processus semblables à l’apoptose et le stress oxidatif. Une meilleure compréhension de l’origine de ces cassures d’ADN mènerait à des études approfondies concernant l’instabilité génétique et le potentiel mutagène induit par le remodelage de la chromatine.

Post-Meiotic DNA Damage and Response in Male Germ Cells

Spermatids are haploid cells that differentiate into spermatozoa and may be considered as an interesting model of DNA damage response and repair. Key features, such a unique set of chromosomes, radioresistance to apoptosis, the presence of known end-joining DNA repair pathways and an underlying prerogative to limit the transmission of any mutation to the next generation, make them a unique cell type to provide new insights on similar pathways in somatic cells. Although DNA damage signaling and repair mechanisms have been extensively studied during meiosis, the contribution of post-meiotic germ cells to the genetic integrity of the male gamete have been overlooked. In this chapter we present clear evidences that the haploid phase of spermatogenesis, termed spermiogenesis, may represent an even greater challenge for the maintenance of the genetic integrity of the male gamete. Since transient DNA strand breaks are intrinsic to the differentiation program of spermatids (Leduc et al., 2008a; Marcon and Boissonneault, 2004), a better understanding of DNA repair pathways involved may shed some light on their potential contribution to male-driven de novo mutations and eventually to some unresolved cases of male infertility. This chapter will mainly focus on DNA breaks occurring in the post-meiotic phase of the spermatogenesis and how germ cells deal with it.

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

The DNA double-strand “breakome” of mouse spermatids

De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.