Localized Delivery of Amifostine Enhances Salivary Gland Radioprotection.

Radiotherapy for head and neck cancers commonly causes damage to salivary gland tissue, resulting in xerostomia (dry mouth) and numerous adverse medical and quality-of-life issues. Amifostine is the only Food and Drug Administration-approved radioprotective drug used clinically to prevent xerostomia. However, systemic administration of amifostine is limited by severe side effects, including rapid decrease in blood pressure (hypotension), nausea, and a narrow therapeutic window. In this study, we demonstrate that retroductal delivery of amifostine and its active metabolite, WR-1065, to murine submandibular glands prior to a single radiation dose of 15 Gy maintained gland function and significantly increased acinar cell survival. Furthermore, in vivo stimulated saliva secretion was maintained in retrograde-treated groups at levels significantly higher than irradiated-only and systemically treated groups. In contrast to intravenous injections, retroductal delivery of WR-1065 or amifostine significantly attenuated hypotension. We conclude that localized delivery to salivary glands markedly improves radioprotection at the cellular level, as well as mitigates the adverse side effects associated with systemic administration. These results support the further development of a localized delivery system that would be compatible with the fractionated dose regimen used clinically.

Salivary Glands: Stem Cells, Self-duplication, or Both?

Understanding the intrinsic potential for renewal and regeneration within a tissue is critical for the rational design of reparative strategies. Maintenance of the salivary glands is widely thought to depend on the differentiation of stem cells. However, there is also new evidence that homeostasis of the salivary glands, like that of the liver and pancreas, relies on self-renewal of differentiated cells rather than a stem cell pool. Here, we review the evidence for both modes of turnover and consider the implications for the process of regeneration. We propose that the view of salivary glands as postmitotic and dependent on stem cells for renewal be revised to reflect the proliferative activity of acinar cells and their role in salivary gland homeostasis.

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.

The molecular biology of Oct-4 in the early mouse embryo.

Preimplantation development in the mouse is characterized by the occurrence of several critical genetic and epigenetic events. Until recently, very little was known about the regulation of these events. The search for genes which are involved in the control of the earliest stages of mouse development has so far resulted in only a few candidates. Oct-4, a member of the POU transcription factor family, is encoded by a gene belonging to this group. Initially present as a maternal factor in the oocyte, Oct-4 is expressed by the embryo throughout the preimplantation period, as well as in germ cell precursors of adult mice. Oct-4 expression is correlated with an undifferentiated phenotype, both in the embryo and in cell lines derived from it. Regulation of the Oct-4 gene is dependent on the activity of two separate enhancers, one of which is specifically active in pluri- and totipotent cells. Its function as a transcriptional regulator is supported by the identification of an increasing number of potential target genes, including some known to be essential for early embryonic development.

A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2-/- results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

Oct-4: more than just a POUerful marker of the mammalian germline?

Mammals lack visible cytoplasmic components in the oocyte that could account for 'germline determinants' as identified in various non-mammalian species. Actually, mammals might not define the germline autonomously by localized 'germline determinants' but conditionally depending on the position of cells within the embryo. The Oct-4 gene encodes a transcription factor that is specifically expressed in the toti- and pluripotential stem cells of the mouse embryo and so far has only been found in mammalian species. Oct-4-expressing embryonal cell retain the capacity to differentiate along multiple lineages and they have been suggested to be part of a 'totipotent germline cycle' that links one generation to the next.

Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles.

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post-natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3(-/- )mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation-contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).

Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells.

The totipotent stem cells of the pregastrulation mouse embryo which give rise to all embryonic somatic tissues and germ cells express Oct-4. The expression is downregulated during gastrulation and is thereafter only maintained in the germline lineage. Oct-4/lacZ transgenes were used to determine how this pattern of expression was achieved, and resulted in the identification of two separate regulatory elements. The distal element drives Oct-4 expression in preimplantation embryos, in migratory and postmigratory primordial germ cells but is inactive in cells of the epiblast. In cell lines this element is specifically active in embryonic stem and embryonic germ cells. The proximal element directs the epiblast-specific expression pattern, including downregulation during gastrulation; in cell lines its activity is restricted to epiblast-derived cells. Thus, Oct-4 expression in the germline is regulated separately from epiblast expression. This provides the first marker for the identification of totipotent cells in the embryo, and suggests that expression of Oct-4 in the totipotent cycle is dependent on a set of factors unique to the germline.

Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6-phosphate signal by two receptors: the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD-MPR-deficient mice with Thp mice that carry a CI-MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI-MPR, like those lacking the CD-MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6-phosphate binding proteins in mammalian cells.

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.

Bone and haematopoietic defects in mice lacking c-fos.

The proto-oncogene c-fos is the cellular homologue of v-fos originally isolated from murine osteosarcoma. Fos protein is a major component of the AP-1 transcription factor complex, which includes members of the jun family. Stable expression of c-fos in mice has been demonstrated in developing bones and teeth, haematopoietic cells, germ cells and in the central nervous system. It has been proposed that c-fos has an important role in signal transduction, cell proliferation and differentiation. We have previously demonstrated that overexpression of c-fos in transgenic and chimaeric mice specifically affects bone, cartilage and haematopoietic cell development. To understand better the function of c-fos in vivo, we used gene targeting in embryonic stem cells to generate cells and mice lacking c-fos. Here we report that heterozygous fos +/- mice appear normal, although females exhibit a distorted transmission frequency. All homozygous fos -/- mice are growth-retarded, develop osteopetrosis with deficiencies in bone remodelling and tooth eruption, and have altered haematopoiesis. These data define the c-Fos protein as an essential molecule for the development of specific cellular compartments.

Fat feeding stimulates only one of the two mRNAs encoding rat intestinal membranous and secreted alkaline phosphatase.

We have identified two mRNAs in rat intestinal mucosa by Northern blot analysis, using cloned cDNAs encoding human placental alkaline phosphatase (PLAP). Probes from both the NH2- and COOH-terminal ends of the human PLAP coding region identified, in rat intestine (especially duodenum), an mRNA of nearly identical size (3 kb) to that found in human placenta. A smaller mRNA (2.7 kb), detected only with the COOH-terminal probe, was more prevalent in jejunum. Following feeding of triacylglycerols, the prevalence of the 2.7 kb mRNA increased over 2-fold. The tissue distribution and response of the 2.7 kb mRNA to fat feeding corresponds exactly with the known behavior of the secreted alkaline phosphatase.

Expression of different-sized placental alkaline phosphatase mRNAs in placenta and choriocarcinoma cells.

The expression of human placental-type alkaline phosphatase (ALPase) in the placenta and in three choriocarcinoma cell lines was examined by translation in vitro and RNA blot analysis using a cDNA for placental ALPase. Placental RNA directed the synthesis of two polypeptides that could be immunoprecipitated with antiserum to placental ALPase. Translation of RNA from the choriocarcinoma cell lines, with or without sodium butyrate treatment, yielded a single immunoprecipitable product of molecular weight intermediate between those of the products from the placenta mRNA. Two cDNA clones for placental ALPase were isolated by antibody screening of a placental cDNA library constructed in lambda gt11. The overlapping cDNAs include 462 nucleotides of coding sequence. RNA blot analysis has confirmed that induction of placental-type ALPase levels during placental development is accompanied by an increase in steady-state placental ALPase mRNA concentrations. Examination of the mRNAs revealed a placental ALPase mRNA of 3.0 kilobases (kb) and a distinct choriocarcinoma placental-type ALPase mRNA of 2.6 kb, implying that transformation of normal to malignant trophoblast is associated with the expression of a distinct placental-type ALPase gene transcript and its protein product.

The beta subunit of human chorionic gonadotropin is encoded by multiple genes.

Two recombinant phage clones bearing sequences corresponding to the beta subunit of human chorionic gonadotropin (hCG beta) were isolated from a human genomic library. The beta sequences were mapped by blot hybridization of restriction digests of these phage DNAs and the nonoverlapping inserts were subcloned in pBR322 and sequenced. The nucleotide-sequencing data show that the hCG beta subunit is encoded by at least three nonallelic genes. Moreover, based on restriction analyses of human placental DNA, these genes may be linked in a single cluster with four other hCG beta-like genes. The sequenced genes all differ in their 5' flanking regions, and none of them is completely homologous in sequence to either of two hCG beta cDNA clones used here. In the translated region of one of these genes, three base substitutions result in two changes from the reported amino acid sequence. In the family of beta-containing glycoprotein hormones, the hCG beta subunit is unique in that it contains an extension of 29 amino acids at its COOH end. The DNA sequence corresponding to this region in the sequenced genes is part of a larger exon. These data show that the COOH-terminal extension does not result from splicing of the primary RNA transcript.

Structure and developmental expression of the chick alpha-actin gene.

Recombinant DNA clones containing chick alpha-actin mRNA sequence have been isolated and used as probes to analyze the structure and developmental expression of the chick alpha-actin gene. The full length, 2000 nucleotide alpha-actin mRNA is detected in poly(A) RNA at early and late stages of in vivo leg muscle development. As expected, the alpha-actin mRNA is present at very low levels at early myogenic stages but is a high abundance species in terminally differentiated muscle. However, most of the alpha-actin mRNA from fused leg muscle is shorter than 2000 nucleotides, and occurs in relatively discrete size classes. An alpha-actin-like mRNA can be detected in poly(A) RNA from early embryonic brain, indicating that transcription of the alpha-actin gene may not be strictly muscle-specific at all stages of development. We have identified at least 3, very short (< 100 base pairs) intervening sequences in the alpha-actin gene which was isolated from a chick genomic library. The structure of the chick alpha-actin gene differs, therefore, from the structures of actin genes from yeast and Drosophila, both of which contain a single, relatively long, intervening sequence.

Molecular cloning of developmentally regulated, low-abundance mRNA sequences from embryonic muscle.

To study the role of low-abundance, embryonic muscle-specific gene transcripts, we have developed a method to screen cDNA clones from embryonic muscle for such sequences. The protocol involves two stages: first, partial enrichment for cDNA clones carrying possible embryo-specific sequences by selecting clones of low-abundance sequences; and second, determination, by hybridization to RNA attached to diazobenzyloxymethyl-paper, which sequences from this category are regulated in an embryonic muscle-specific manner during development. At least three different clones were obtained which hybridized to sequences present in early muscle development but absent, or present at relatively low levels, at late embryonic and adult muscle stages. Two of these clones were not muscle-specific because they hybridized to poly(A)+RNA from liver or brain or both. The third clone, 106A4, did not detectably hybridize to total poly(A)+RNA at any stage of brain or liver development tested. This sequence also was not detectable in poly(A)+RNA from embryonic muscle progenitor cells. Thus, the 106A4 sequence is a likely candidate for an embryonic muscle-specific sequence. We have demonstrated that the 106A4 sequence is a mRNA, although the specific identity and function of the translated product is unknown. The method used to identify embryonic muscle-specific cDNA clones should be generally applicable for obtaining clones for low abundance transcripts regulated in a tissue-specific or developmental stage-specific manner.