Rectal cancer lateral lymph nodes: multicentre study of the impact of obturator and internal iliac nodes on oncological outcomes.

BACKGROUND: In patients with rectal cancer, enlarged lateral lymph nodes (LLNs) result in increased lateral local recurrence (LLR) and lower cancer-specific survival (CSS) rates, which can be improved with (chemo)radiotherapy ((C)RT) and LLN dissection (LLND). This study investigated whether different LLN locations affect oncological outcomes. METHODS: Patients with low cT3-4 rectal cancer without synchronous distant metastases were included in this multicentre retrospective cohort study. All MRI was re-evaluated, with special attention to LLN involvement and response. RESULTS: More advanced cT and cN category were associated with the occurrence of enlarged obturator nodes. Multivariable analyses showed that a node in the internal iliac compartment with a short-axis (SA) size of at least 7 mm on baseline MRI and over 4 mm after (C)RT was predictive of LLR, compared with a post-(C)RT SA of 4 mm or less (hazard ratio (HR) 5.74, 95 per cent c.i. 2.98 to 11.05 vs HR 1.40, 0.19 to 10.20; P < 0.001). Obturator LLNs with a SA larger than 6 mm after (C)RT were associated with a higher 5-year distant metastasis rate and lowered CSS in patients who did not undergo LLND. The survival difference was not present after LLND. Multivariable analyses found that only cT category (HR 2.22, 1.07 to 4.64; P = 0.033) and margin involvement (HR 2.95, 1.18 to 7.37; P = 0.021) independently predicted the development of metastatic disease. CONCLUSION: Internal iliac LLN enlargement is associated with an increased LLR rate, whereas obturator nodes are associated with more advanced disease with increased distant metastasis and reduced CSS rates. LLND improves local control in persistent internal iliac nodes, and might have a role in controlling systemic spread in persistent obturator nodes.Members of the Lateral Node Study Consortium are co-authors of this study and are listed under the heading Collaborators.

Prognostic implications of MRI-detected lateral nodal disease and extramural vascular invasion in rectal cancer.

BACKGROUND: Lateral nodal disease in rectal cancer remains a subject of debate and is treated differently in the East and the West. The predictive value of lateral lymph node and MRI-detected extramural vascular invasion (mrEMVI) features on oncological outcomes was assessed in this study. METHODS: In this retrospective cohort study, data on patients with cT3-4 rectal cancer within 8 cm from the anal verge were considered over a 5-year period (2009-2013). Lateral lymph node size, malignant features and mrEMVI features were evaluated and related to oncological outcomes. RESULTS: In total, 192 patients were studied, of whom 30 (15·6 per cent) underwent short-course radiotherapy and 145 (75·5 per cent) received chemoradiotherapy. A lateral lymph node short-axis size of 10 mm or more was associated with a significantly higher 5-year lateral/presacral local recurrence rate of 37 per cent, compared with 7·7 per cent in nodes smaller than 10 mm (P = 0·041). Enlarged nodes did not result in a higher 5-year rate of distant metastasis (23 per cent versus 27·7 per cent in nodes smaller than 10 mm; P = 0·563). However, mrEMVI positivity was related to more metastatic disease (5-year rate 43 versus 26·3 per cent in the mrEMVI-negative group; P = 0·014), but not with increased lateral/presacral recurrence. mrEMVI occurred in 46·6 per cent of patients with nodes smaller than 10 mm, compared with 29 per cent in patients with nodes of 10 mm or larger (P = 0·267). CONCLUSION: Although lateral nodal disease is more a local problem, mrEMVI mainly predicts distant recurrence. The results of this study showed an unacceptably high local recurrence rate in patients with a short axis of 10 mm or more, despite neoadjuvant (chemo)radiotherapy.

Inhibition of HIF-1alpha by the anticancer drug TAS106 enhances X-ray-induced apoptosis in vitro and in vivo.

In a previous study, we showed that a novel anticancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (TAS106, ECyd) increased the antitumour efficacy of X-irradiation. However, its effects on hypoxic cells in tumours remain unclarified. Here, we show that TAS106 enhances the induction of apoptosis in X-irradiated human gastric adenocarcinoma MKN45 and MKN28 cells under hypoxia in vitro. At the same time, the accumulation of HIF-1alpha observed under hypoxia was shown to be decreased to the level of normoxia in the presence of 0.1 microM TAS106. To study the function of HIF-1alpha protein in apoptosis of hypoxic cells, we employed an HIF-1alpha reductive approach using its specific antisense oligodeoxynucleotide. The reduction of HIF-1alpha gene expression dramatically enhanced X-ray-induced apoptosis in hypoxic cells. In in vivo experiments in which MKN45 cells were transplanted into severe combined immunodeficient (SCID) mice, TAS106 (0.5 mg kg(-1)) suppressed HIF-1alpha expression and subsequently reduced the area of the hypoxic region in the tumour and enhanced the induction of apoptosis in the hypoxic region when combined with 2 Gy of X-irradiation. These results suggest the possibility that TAS106 acts as a potent radiosensitiser through the inhibition of HIF-1alpha expression and can be a useful agent against radiotherapy-resistant hypoxic cells in solid tumours.

Correction of a genetic defect in multipotent germline stem cells using a human artificial chromosome.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53-/- mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.

Molecular characterization of the developmental gene in eyes: through data-mining on integrated transcriptome databases.

OBJECTIVES: Our aim was to utilize publicly available and proprietary sources to discover candidate genes important for ocular development. DESIGN AND METHODS: The collated information on our 5092 non-redundant clusters was grouped and functional annotation was conducted using gene ontology (FatiGO) for categorizing them with respect to molecular function. The web-based viewer technological platform (H-InvDB) was employed for transcription analyses of in-house high quality fetal eye Expressed Sequence Tags (ESTs). Eye-specific ESTs were also analyzed across species by using EMBEST. RESULTS: According to adult eye cDNA libraries, nucleic acid binding and cell structure/cytoskeletal protein genes were the most abundant among the ESTs of fetal eyes. Using cDNA assembly in H-InvDB, 20 (80%) of the 25 most commonly expressed genes in the human eye are also expressed in extraocular tissues. The crystalline gamma S gene is highly expressed in the eye, but not in other tissues. We used EMBEST to compare human fetal eye and octopus eye ESTs and the expression similarity was low (1.6%). This indicated that our fetal eye library contains genes necessary for the developmental process and biological function of the eye, which may not be expressed in the fully developed octopus eyes. The human fetal eye cDNA library also contained highly abundant eye tissue genes, including alphaA-crystallin, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), bestrophin (VMD2), cystatin C, and transforming growth factor, beta-induced (BIGH3). CONCLUSIONS: Our annotated EST set provides a valuable resource for gene discovery and functional genomic analysis. This display will help to appreciate the strengths and weaknesses of the different technological platforms, so that in future studies the maximum amount of beneficial information can be derived from the appropriate use of each method.

Germline niche transplantation restores fertility in infertile mice.

BACKGROUND: Stem cells interact closely with their microenvironment or niche, and abnormalities in niche compromise the self-renewing tissue. In testis, for example, Sertoli cells interact with germ cells, and defects in Sertoli cells compromises spermatogenesis, leading to male infertility. However, it has not been possible to restore spermatogenesis from endogenous stem cells in infertile testis with environmental defects. METHODS AND RESULTS: When healthy Sertoli cells from infertile white spotting (W) mouse were transplanted into the seminiferous tubules of infertile Steel (Sl) mouse testis that had defective Sertoli cells, spermatogenesis occurred from Sl stem cells in the recipient testis. On average, 1.1% of the recipient tubules showed spermatogenesis. Furthermore, in a microinsemination experiment with germ cells that developed in the testis, we obtained four normal offspring from 114 successfully injected oocytes. CONCLUSIONS: This study demonstrates that defects in male germline microenvironment can be corrected by Sertoli cell transplantation. Although further improvements are required to enhance the low efficiency of spermatogenesis, the ability to correct environmental defect by niche transplantation has important implications in developing new strategies for treating incurable disorders in self-renewing tissues.

Augmented cytoplasmic Smad4 induces acceleration of TGF-beta1 signaling in renal tubulointerstitial cells of hereditary nephrotic ICGN mice with chronic renal fibrosis; possible role for myofibroblastic differentiation.

The Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse is a hereditary model animal for nephrotic syndrome with chronic renal tubulointerstitial fibrosis. In most fibrotic diseases, myofibroblastic differentiation is considered to play crucial roles in pathogenesis of fibrosis and is dominantly regulated by the transforming growth factor (TGF)-beta1 signaling system. To reveal the pathogenic mechanism of chronic renal fibrosis in ICGN mice, we examined the expression and localization of TGF-beta1 signal transducer proteins (TGF-beta receptor-I and -II, Smad2/3 and Smad4) in kidney sections and in primarily cultured tubulointerstitial fibroblasts (TIFs). In kidneys of ICGN mice, many tubulointerstitial cells were differentiated to myofibroblastic cells and were alpha-smooth muscle actin (alphaSMA)-positive. The numbers of alphaSMA-positive TIFs prepared from kidneys of ICGN mice (ICGN-TIFs), but not those of ICR control mice (ICR-TIFs), increased during cell culture. No significant differences in production or activation of TGF-beta1 between ICGN-TIFs and ICR-TIFs were seen by enzyme-linked immunosorbent assay. In vitro transcriptional reporter assay for TGF-beta1 and Western immunoblotting for TGF-beta1 signal transducers showed no notable differences in the expression levels of TGF-beta receptor-I or -II or Smad2/3 between these TIFs. However, augmented cytoplasmic Smad4 protein in ICGN-TIFs, but not ICR-TIFs, seemed to cause hypersensitivity against TGF-beta1, and the eventual nuclear localization of Smad2/3-Smad4 complex was increased in ICGN-TIFs. Thus, the abnormal cytoplasmic augmentation of Smad4 induces acceleration of TGF-beta1 signaling in the renal tubulointerstitial cells of ICGN mice.

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells.

BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.

Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination.

BACKGROUND: Fertility protection is an urgent clinical problem for prepubertal male oncology patients who undergo either chemotherapy or radiotherapy. As these patients do not have mature sperm to be frozen, there is as yet no effective method to preserve their fertility. METHODS AND RESULTS: Single pieces of immature mouse (1.5 x 1.5 x 1.5 mm) or rabbit (2.0 x 2.0 x approximately 3.0 mm) testis were cryopreserved, thawed and transplanted into mouse testes. Histological techniques were used to determine the presence of spermatogenesis, which was restored in both mouse and rabbit testicular pieces, and led to the production of mature sperm after both cryopreservation and syngeneic or xenogeneic transplantation into mouse testes. Using sperm developed in the frozen-thawed transplants, mouse offspring were born after in-vitro microinsemination. Furthermore, rabbit offspring were obtained using rabbit sperm that developed in fresh transplants in a xenogeneic surrogate mouse. CONCLUSIONS: This approach of 'testicular tissue banking' is a promising technique for the preservation of fertility in prepubertal male oncology patients. Xenogeneic transplantation into immunodeficient mice may provide a system for studying spermatogenic failure in infertile men.

Neurotoxic and neuroprotective effects of glutamate are enhanced by introduction of amyloid precursor protein cDNA.

The physiological role of amyloid precursor protein (APP), whose anomalous metabolite is a putative pathogen for Alzheimer disease, remains unclear. From the enhanced responsiveness to glutamate in cultured hippocampal neurons after the introduction of cDNA of APP695 (an isoform of APP dominant in human brain) using an adenovirus vector, we have recently raised the hypothesis that APP modulates neuronal sensitivity to glutamate. To test this hypothesis, we utilized here the unique effects of glutamate on the survival of different types of neurons. It is known that hippocampal neurons undergo deterioration in 24 h after application of glutamate in a dose-dependent manner. This vulnerability was increased in the cells transfected with adenovirus carrying cDNA of APP695. By contrast, it is known that cerebellar granule neurons require for their survival the supplementation of NMDA to the medium. The dose of NMDA required for survival was reduced after the transfection of the APP-adenovirus to cerebellar granule neurons. These enhancing effects of APP on the glutamate-induced vulnerability in hippocampal neurons and the glutamate (NMDA)-dependent survival in cerebellar neurons were blocked by glutamate receptor inhibitors, and were not seen after application of a control adenovirus carrying cDNA of beta-galactosidase. Since the effects of glutamate were enhanced in both directions, the hypothesis became more likely that one of the physiological functions of cellular APP is the regulation of glutamate receptors.

Increase in number of functional release sites by cyclic AMP-dependent protein kinase in cultured neurons isolated from hippocampal dentate gyrus.

The enhancement of synaptic exocytosis is one form of long-term potentiation (LTP) of synaptic transmission. As possible mechanisms underlying this enhancement, increases in the release probability and/or the number of release sites are suggested. To obtain direct evidence for the increase in the number of functional release sites induced by protein kinase A (PKA) cascade, we attempted to visualize functional release sites using styryl dyes, FM4-64 and FM1-43, and investigated the effects of PKA on the release sites. A PKA activator FSK increased the number of active release sites by approximately 20-30%. A direct PKA activator, Sp-cAMPS, showed the same effect, which was blocked by a PKA inhibitor, KT5720, suggesting that this effect was mediated by PKA. This PKA-dependent increase in the number of release sites requires Ca(2+) in the bath solution, and Sr(2+) can not be a substitute for Ca(2+). Since the number of functional release sites is approximately half the total number of synaptophysin-immunoreactive sites, the PKA dependent activation of silent release sites of DG neuron terminals is suggested.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

Development of embryos in superovulated guinea pigs following active immunization against the inhibin alpha-subunit.

Embryo recovery and subsequent embryonic development from guinea pigs treated with or without inhibin vaccines were compared to determine the effect of active immunization against the inhibin alpha-subunit. Twenty female guinea pigs of the Hartley strain were injected 3 times either with 1 ml inhibin vaccine (recombinant ovine inhibin a-subunit in oil emulsion: 50 microg/ml, inhibin-immunized group), or 1 ml placebo (saline in oil emulsion; control group) at 4 week intervals. After one estrous cycle following the last injection, females were naturally mated and embryos were collected at 11:00 hr of day 6 of pregnancy (Day 1: sperm in the vaginal smear) for culture in vitro. Active immunization increased the number of corpora lutea (12.6+/-3.0 vs. 4.6+/-0.2, P<0.05), recovered embryos (9.8+/-1.9 vs. 3.6+/-0.4, P<0.01) and normal embryos (7.8+/-1.4 vs. 3.6+/-0.4, P<0.05), although estrous cycle length was not affected (P>0.05). During subsequent 8 day culture in vitro, most of the recovered embryos formed trophoblast outgrowth; 100% (14/14) and 88.2% (15/17) in control and immunized groups, respectively. High levels of inhibin antibody titers were sustained in the inhibin-immunized guinea pigs at least for 5 months after the last injection while no antibody titer was detected in the control animals. These results indicate that active immunization against the inhibin a-subunit is a long-acting and efficient method to induce superovulation with normal embryonic development in the guinea pig.

Decreased matrix metalloproteinase activity in the kidneys of hereditary nephrotic mice (ICGN strain).

Abnormalities of extracellular matrix (ECM) metabolism, i.e., overproduction and/or inhibition of ECM breakdown, may contribute to progression of fibrotic degeneration in the kidney. Earlier studies revealed that major ECM components, type I, III, and IV collagens, etc., were accumulated in glomeruli and tubulointerstitium in kidneys of Institute of Cancer Research (ICR) derived glomerulonephritis (ICGN) mice which are a novel inbred strain of mice with a hereditary nephrotic syndrome of unknown etiology and are considered to be a good model of human idiopathic nephrotic syndrome. In the present study, we compared the activities of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components, in the kidneys of aged ICGN mice and age-matched ICR mice as normal controls. We biochemically measured interstitial collagenase (MMP-1), gelatinase (MMP-2 and MMP-9), and stromelysin (MMP-3) activities in the kidney tissues. Lower activities of MMP-1 and MMP-2 and MMP-9 were demonstrated in the kidneys of ICGN mice as compared with those of ICR mice, but there were no significant differences in the MMP-3 activities between these strains. These results show that decreased MMP activities cause abnormal accumulation of ECM in ICGN mouse kidneys.

Birth of mice after nuclear transfer by electrofusion using tail tip cells.

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.

The postmitotic growth suppressor necdin interacts with a calcium-binding protein (NEFA) in neuronal cytoplasm.

Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca(2+)-binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca(2+) binding activity as determined by (45)Ca(2+) overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca(2+) levels. Thus, necdin and NEFA might be involved in Ca(2+) homeostasis in neuronal cytoplasm.

Follicle selection in cyclic guinea pigs with active immunization against inhibin alpha-subunit.

Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine alpha-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3+/-0.3 to 7.0+/-1.6 per each ovary), and preovulatory sized follicles (from 1.8+/-0.6 to 7.0+/-1.6 per each ovary), and follicles stained positively for inhibin alpha-subunit (from 2.3+/-0.5 to 6.3+/-1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin alpha-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.

Ras mediates effector pathways responsible for pre-B cell survival, which is essential for the developmental progression to the late pre-B cell stage.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.