Id (inhibitor of differentiation or DNA binding) gene encodes a helix-loop helix protein which dimerizes and blocks the basic HLH protein from binding DNA. It expresses mainly in actively dividing cells and was first reported to be involved in hormone-induced prostate cancer in the Noble rat model. It was subsequently confirmed also in human prostate cancer. Through functional studies under in vitro system, they have further demonstrated the role of Id1 in prostate cancer progression. They have shown that Id1 transfection stimulated prostate cancer cell proliferation through downregulation of p16/Rb, while inducing activation of MAPK and NFκB pathways. Activation of the latter pathways by ectopic transfection of Id1 to LNCaP cells, an androgen dependent line, also resulted in reduced sensitivity of prostate cancer cells to androgen on the one hand and upregulating the expression of EGFR and PSA on the other hand, which are both hallmarks of androgen independent prostate cancer. This review shows the crucial role played by Id1 in the conversion of prostate cancer from androgen dependent to androgen independent stage.
Common immunohistochemistry is a technique based on antigen-antibody reactions in thin sections. It is well known that the important steps of tissue preparation protocols are (i) fixation (chemical and physical methods, especially cryofixation), (ii) sectioning, and (iii) immunostaining. We have already examined how to combine these preparation steps for an improved immunohistochemistry of target cells and tissues so as to overcome some of the technical problems commonly encountered. In many experimental cases, various cryotechniques were useful to obtain both a strong immunoreactivity of the antigens and a high time-resolution of the morphology of living animal cells and tissues. When the ultimate aim is to obtain in vivo observation of the morphology and immunolocalization, the new concept of in vivo cryotechnique becomes indispensable in the field of cell biology.
Visualizing how signals are transmitted within a living cell has long been a goal of molecular biologists, which has now been realized by probes based on the principle of fluorescence resonance energy transfer (FRET). Variants of green fluorescent protein (GFP) enabled the preparation of genetically-encoded FRET probes, and their application has been expanded for use in many areas of biology. The GFP-based FRET probes can be classified as belonging to one of two types, intermolecular and intramolecular FRET probes. The merit of the intermolecular FRET probe lies in the ease of preparation of the probes, whereas the merit of the intramolecular FRET probe lies in the high signal-to-noise ratio. Although these GFP-based probes are powerful tools for the visualization of signal transduction cascades, numerous pitfalls remain associated with this technique. Here, we provide an overview of the GFP-based FRET probes and discuss these issues.
The quick-freezing method often used for physical fixation also contributed enormously to the advancement of morphology, but it could not provide enough information about the dynamic morphological changes in vivo in living animal organs. Therefore, we developed the in vivo cryotechnique in 1995, which could directly cryofix organs in vivo under anesthetized conditions without stopping blood circulation or producing effects of anoxia. We have already reported new findings about the in vivo ultrastructures of living animal organs with the cryotechnique followed by freeze-substitution or replica preparation. Recently, it has also been applied to other morphological analyses in vivo, including immunohistochemistry and FISH, for obtaining dynamically functioning structures of cells and tissues at a light microscopic level. In such experimental processes, the in vivo cryotechnique has been shown to have the added benefit of direct antigen-retrieval effects since it reduces several steps of retrieval treatments which are required in common paraffin-embedded samples prepared by the conventional fixation and dehydration. In conclusion, the in vivo cryotechnique allows us to investigate the functioning real morphology of living animals, which was technically difficult to be observed before, and perform dynamic immunohistochemistry of cells and tissues in various animal organs in vivo at ultimate time-resolution.
Spermatogenic immunoglobulin superfamily (SgIGSF) is an adhesion molecule originally isolated from adult mouse testis. In the testis, SgIGSF is expressed specifically in spermatogenic cells and may be involved in spermatogenesis. SgIGSF may also be involved in synapse formation and tumor suppression. In the present study, we examined the expression and cellular localization of SgIGSF in the entire adult mouse organs and tissues using Western blot analysis and immunohistochemistry at light and electron microscopic levels. Western blot analysis revealed that SgIGSF is expressed not only in the testis but also in the liver, lung, and nervous system including the cerebrum, cerebellum and sciatic nerve. The nervous system as well as testis showed multiple immunoreactive bands ranging from 45 to 100 kDa, whereas the liver and lung showed a single 100 kDa band. Immunohistochemistry demonstrated that in the nervous system, SgIGSF is localized to the membranes of synapses, axons and Schwann cells. In contrast, in the lung and liver SgIGSF was localized to the membranes of apposing respiratory epithelial cells, hepatocytes and biliary epithelial cells. These results suggested that SgIGSF plays multiple physiological roles in the adult mouse as an adhesion molecule.
Caveolin-3 is thought to serve as a structural and a scaffolding protein in the caveolae of striated muscle. The present study aims to clarify the precise localization of caveolin-3 and its interactive protein partners in adult murine cardiac muscle by immunohistochemistry, immunoblot and immunoprecipitation. Double labeling confocal images showed that caveolin-3 is colocalized with dihydropyridine receptor α1, ryanodine receptor, and α-actinin, but not sarcoplasmic or endoplasmic reticulum calcium ATPase 2. Immunoelectron microscopy demonstrated that caveolin-3 is localized at the T-tubules. Subsequently, we investigated the protein interaction between caveolin-3 and α-actinin, a major component of the Z-band, using immunoprecipitation and immunoblot. It was of interest to note that caveolin-3 was coprecipitated with α-actinin in cardiac muscle extraction but not in skeletal muscle. Taken together, these data indicate that caveolin-3 serves as a mediator between sarcomeric dynamics via the Z-bands and T-tubule function in cardiac muscle.
Keratinocyte growth factor (KGF), an androgen-dependent epithelial mitogen, and its receptor (KGFR) have been implicated in the regulation of cell growth and differentiation in prostate tissue. This study was designed to determine the expression and role of KGF and KGFR in normal and prostate cancer tissues, especially in relation to cell kinetics. In 41 cases of prostate cancer in paraffin-embedded specimens, the expression of KGF and KGFR at the levels of protein and mRNA was analyzed by immunohistochemistry using newly raised antibodies and in situ hybridization, respectively. We also examined expression of androgen receptor (AR) and Ki-67 labeling index (LI). In normal and hyperplastic prostate tissues, both KGF mRNA and protein were localized in AR positive stromal cells, while those of KGFR were localized in glandular epithelial cells. In prostate cancer, however, coexpression of KGF and KGFR was observed in 14/41 cases, and significantly correlated with high Gleason scores, bone metastasis and high Ki-67 LI. The relapse-free survival of patients suffering from prostate cancers coexpressing KGF and KGFR was significantly shorter than that of patients from the other ones. Therefore, our results indicate that coexpression of KGF and KGFR in prostate cancer may predict metastatic and proliferative activities, possibly due to the formation of an autonomous andromedin loop.
To understand proliferation and suppression in ameloblastoma that exhibit locally invasive growth and recur clinically, the expression of proliferating cell nuclear antigen (PCNA), Ki-67 antigen, topoisomerase IIα, p53 and p21 proteins were analyzed with immunohistochemistry. Sections of archival ameloblastoma tissues were used (16 plexiform and 14 follicular types). Each antigen was labeled by the polymer method with optimal antigen retrieval. The plexiform type exhibited higher expression of PCNA, Ki-67 and topoisomerase IIα antigens than the follicular type. Expression of PCNA correlated significantly with that of the Ki-67 antigen. The plexiform type also exhibited greater expression of p53 and p21 proteins than the follicular type. Expression of p53 protein correlated significantly with that of p21, PCNA, Ki-67 antigen and topoisomerase IIα. Expression of p21 protein did not correlate with that of the proliferation-related antigens. These findings suggest that the plexiform ameloblastoma has higher proliferating activity and malignant potentiality as neoplastic cells than the follicular ameloblastoma.
The degeneration of organelles in the course of keratinization and in other terminal differentiation pathways has been studied mainly by electron microscopy. Here I show a panoramic view of the degeneration of ribosomes in sections of the skin epidermis, hair shaft and hair inner root sheath of neonatal rats by in situ hybridization using a complementary DNA probe for 28S-rRNA. As the keratinization advances, staining for 28S-rRNA decreases reciprocally. In contrast, immunohistochemical signals of RNase A increase in the spinous layers of the epidermis as well as in the corresponding differentiation stage of the inner root sheath of the hair, suggesting the contribution of this enzyme to the degeneration of ribosomes in these areas. In the hair shaft, however, RNase A was hardly detectable in any differentiation stage. RNase A is extraordinarily rich in the outer root sheath, which does not undergo keratinization, while 28S-rRNA was detected faintly in this tissue. Since the outer root sheath is thought to be a barrier to pathogens, RNase A may function as a weapon against retroviruses. In contrast to the outer root sheath, 28S-rRNA is rich in the basal layer of the epidermis and hair matrix of the hair bulb, while RNase A is poor in these tissues, which are located in the deep skin and undergo active cellular proliferation. Most dermal fibroblasts exhibit strong RNase A signal but weak 28S-rRNA signals. As exceptions, the dermal papilla and dermal sheath showed lower levels of RNase A.
It was long ago reported that human amniotic epithelial cells consisted of two morphologically different cells: one had more abundant Golgi apparatus and mitochondria with more lucent cytoplasm, while the other had sparse intracytoplasmic organelles with darker cytoplasm. We semiquantitatively analysed the number of Golgi apparatus and mitochondria of the human amniotic epithelial cells, employing cytochrome c oxidase enzymehistochemistry as the marker for mitochondria. In the specimens fixed in glutaraldehyde solution, two types of cells with differing degrees of cytoplasmic darkness were observed. However, the number of Golgi apparatus and mitochondria was not different between these two cell types. The biological significance of differing degrees of cytoplasmic darkness seen in the chemically-fixed amniotic epithelial cells may deserve further study.