Sergio A. Lamprecht

Cellular Mechanisms of Calcium and Vitamin D in the Inhibition of Colorectal Carcinogenesis

Convincing evidence is available showing that dietary calcium and vitamin D impede the development of colonic carcinogenesis. The major cellular modes of action of calcium and vitamin D which can contribute to the inhibition of colonic neoplasia are reviewed in this article. These consist of complex series of signaling events induced by the chemopreventive agents acting at various tiers of colonic cell organization.

A protective role of dietary vitamin D3 in rat colon carcinogenesis.

The aim of the present work was to gain insight into a putative anticancer effect of dietary vitamin D3 (cholecalciferol) in a rat model of colon carcinogenesis. Male rats were assigned to three different dietary groups. The dietary regimens were based on a standard murine-defined diet (AIN-76A) or a stress diet containing 20% fat, reduced Ca2+ concentration, a high phosphorus-to-Ca2+ ratio, and either low or high vitamin D3 content. Colorectal cancer was induced by administration of the procarcinogen 1,2-dimethylhydrazine (DMH). Blood Ca2+, 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], and 25-hydroxyvitamin D3 [25(OH)D3 ] levels were measured in DMH-treated rats and in respective weight- and age-matched dietary control groups. Colonic epithelial proliferation was assessed by determining thymidine kinase (TK) activity, bromodeoxyuridine (BrdUrd) incorporation into crypt cell DNA, and the mean labeling index along the colonic crypt continuum. Maintenance of rats on the stress diet either unmodified or supplemented with vitamin D3 in the absence of carcinogen treatment provoked a time-dependent rise in colonic TK activity and hyperproliferation of colonic epithelium. DMH treatment of rats maintained on the standard diet caused a marked increase in the proliferative indexes of colonic epithelium and in expansion of the crypt proliferative compartment. TK activity and the crypt mitotic zone were significantly augmented in the animal group fed the stress diet. Supplementary vitamin D3 abrogated the stress diet-enhanced colonic responses to the carcinogenic insult. Colon tumor multiplicity was fourfold higher in animals fed the stress diet than in animals maintained on a standard diet. The marked rise in colonic tumor multiplicity and adenocarcinoma incidence in rats fed the stress diet was obliterated by supplemental dietary vitamin D3 Cumulatively, the present results indicate that dietary vitamin D3 impedes the neoplastic process in murine large intestine and strengthen the view that inappropriate changes in dietary components and micronutrients are contributory determinants of colorectal cancer.

Isolation and characterization of normal and neoplastic colonic epithelial cell populations.

The aim of the present study was to characterize rat mucosal colonic cells harvested from the crypt continuum during differentiation and dimethylhydrazine-induced neoplasia. The collection of colonocytes was performed using a modified nonenzymatic isolation procedure based on Ca2+ chelation and gentle mechanical dissociation. Light and electron microscopy histomorphological examinations, [3H]thymidine incorporation studies, and activity gradients of alkaline phosphatase, thymidine kinase, and cytoskeleton-associated protein tyrosine kinase indicated that distinct cell populations were harvested from the various crypt regions in a temporal sequence mirroring their zonal and functional distribution in situ. After dimethylhydrazine administration, marked protein tyrosine kinase activity was noted in colonic cells harvested from upper crypt zones. The misplaced and sustained kinase activity preceded the actual polyp or tumor formation. This observation is consistent with the expansion of colonic proliferative compartments beyond allowable boundaries during the preneoplastic period. Companion studies in human colonic epithelial specimens corroborate the findings observed in normal and transformed murine colonocytes. It is believed that the characterization and manipulation of colonocytes using our in vitro model will provide important clues to the molecular events underlying the differentiation program and carcinogenic process in the colonic cell.

Apoptosis (programmed cell death) in colonic cells: from normal to transformed stage

We determined apoptosis in whole rat colonic tissue and in isolated colonocytes from the various rat crypt regions in preneoplastic stages up to frank neoplasia following administration of the procarcinogen, dimethylhydrazine (DMH). Apoptotic cells were determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-method, by evaluating sections stained with hematoxylin and eosin, and caspase-1 immunostaining. Apoptotic cells in whole colonic tissue from untreated rats were confined to the upper crypt while, in DMH-treated rats apoptotic and caspase-1 positive cells were located in the crypt proliferative regions. Numerous apoptotic and caspase-1-positive cells were found in sections from early tumors while in the delayed tumors, apoptotic-positive cells were absent and number of caspase-1-positive cells was negligible. A marked reduction in the apoptotic index along the crypt was observed in isolated transformed colonic cells, this was not the case for caspase-1-positive cells. We conclude that: (i) in colorectal tumors at progressive stage apoptosis is altered, (ii) the mechanistic alteration in apoptosis may be located between caspase-1-protease activity and the fragmentation process of DNA.

Extensive apoptotic death of rat colonic cells deprived of crypt habitat

Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell‐to‐cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD‐CMK) inhibitor of interleukin‐1β‐converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium‐orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic‐labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD‐CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE‐mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype. J. Cell. Physiol. 177:377–386, 1998.

Differential distribution of protein kinases along the crypt-to-lumen regions of rat colonic epithelium.

The activity of cAMP-dependent and cAMP-independent protein kinases, a class of enzymes involved in the regulation of cell proliferation was measured in rat colonic epithelium. Sequential cell populations harvested by a stepwise scraping technique from colonic crypt regions were identified by histology and incorporation of [3H]-thymidine into DNA. cAMP-independent phosphorylation of casein, in the presence of [gamma-32P]ATP, was markedly suppressed by quercetin, a bioflavonoid known to inhibit G-type casein kinase, protein kinase-C and tyrosine protein kinase. Conversely, the cyclic nucleotide regulatable form requiring histone as substrate was responsive to the action of the heat stable protein kinase inhibitor. The protein kinase species were characterised and partially purified by DEAE-cellulose chromatography. The activity of cAMP-dependent protein kinase in colonic cytosols (pmol 32P/min/mg protein, means (SE)) increased from 129.4 (15.9) in superficial cell populations to 238.5 (31.4) in lower crypt cell fractions (p less than 0.01). Colonic cAMP-independent protein kinase activity increased from 87.3 (15.6) in surface cell preparations to 178.1 (30.0) in lower crypt cell populations (p less than 0.02). A comparable activity gradient was observed in membrane fractions. The activity gradient persisted when the results were expressed as a function of cellular DNA. These findings indicate that protein kinases display a defined topological segregation along the colonic crypt regions and that during migration to the lumen colonic cells attenuate enzyme signals supposedly related to tissue growth.

The cancer cells-of-origin in the gastrointestinal tract: progenitors revisited

A prominent model of tumor progression posits that normal self-renewing and multipotent stem cells(SCs) are the initial target of transformation. This view has been robustly challenged by the recurring observation that transit-amplifying cells and differentiated progenitors can initiate neoplasia outside the SC zone thus qualifying as cancer cells-of-origin. The emerging concept is that a cancer SC and a cancer cell-of-origin are not necessarily the same cell. Importantly, progenitor cells were shown to possess remarkable plasticity and to revert, on demand, to a SC-like state. The present review revisits our early hypothesis that colonic progenitors acquiring a mutant adenomatous polyposis coli gene after exiting the stem zone may serve as genuine cancer cells-of-origin. New findings consonant with this view are examined, and tenable molecular and cellular mechanisms underpinning the plasticity of progenitor cells in the gastrointestinal tract and in other tissues are discussed. The translational impact of cell plasticity is addressed, and recommendations for future research are advanced.

Controlled release of TGF-β1 impedes rat colon carcinogenesisin vivo

Transforming growth factor β1 (TGF‐β1) is a cytokine known to play a key role in the control of cell growth. TGF‐β1 potently inhibits the proliferation of human and rodent‐derived epithelial cells. Colonic precancerous and moderately differentiated cancer cells are responsive to TGF‐β1, whereas malignant colon cancer cells are resistant to the inhibitory action of the cytokine. These observations have been derived exclusively from in vitro studies. Therefore, the main aim of our study was to determine whether TGF‐β1 exerts a growth‐restraining action on colon carcinogenesis in vivo. TGF‐β1 was sequestered into ethylene acetate copolymer matrices and “loaded” preparations were implanted intraperitoneally (i.p.) in rats. One week later, the animals were treated with dimethylhydrazine (DMH), a colon procarcinogen. Empty matrices devoid of TGF‐β1 but containing bovine serum albumin (BSA) carrier served as the appropriate control preparations. The number of aberrant crypt foci (ACF), considered to be preneoplastic lesions of the colon, was scored. Tumor formation and size were assessed at the appropriate times. TGF‐β1 released in a sustained manner from copolymer matrices: (i) markedly inhibited colonic ACF formation and the number of aberrant crypts and (ii) significantly reduced colonic tumor formation and size. Int. J. Cancer 78:618–623, 1998.

Cytosolic phospholipase A2 α has a crucial role in the pathogenesis of DSS-induced colitis in mice.

Colitis, an inflammation of the colon, is a well‐characterized massive tissue injury. Cytosolic phospholipase A2α (cPLA2α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA2α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA2α and NF‐κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP‐R14, ICAM‐1, COX‐2, iNOS upregulation and LTB4 and TNF‐α secretion. Prevention of cPLA2α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA2α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF‐κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA2α upregulation in inflammation and development of murine colitis.

Teaming Up for Trouble: Cancer Cells, Transforming Growth Factor-β1 Signaling and the Epigenetic Corruption of Stromal Naïve Fibroblasts

It is well recognized that cancer cells subvert the phenotype of stromal naïve fibroblasts and instruct the neighboring cells to sustain their growth agenda. The mechanisms underpinning the switch of fibroblasts to cancer-associated fibroblasts (CAFs) are the focus of intense investigation. One of the most significant hallmarks of the biological identity of CAFs is that their tumor-promoting phenotype is stably maintained during in vitro and ex vivo propagation without the continual interaction with the adjacent cancer cells. In this review, we discuss robust evidence showing that the master cytokine Transforming Growth Factor-β1 (TGFβ-1) is a prime mover in reshaping, via epigenetic switches, the phenotype of stromal fibroblasts to a durable state. We also examine, in detail, the pervasive involvement of TGFβ-1 signaling from both cancer cells and CAFs in fostering cancer development, taking colorectal cancer (CRC) as a paradigm of human neoplasia. Finally, we review the stroma-centric anticancer therapeutic approach focused on CAFs—the most abundant cell population of the tumor microenvironment (TME)—as target cells.