Olaparib monotherapy as primary treatment in unselected triple negative breast cancer.

BACKGROUND: The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure. PATIENTS AND METHODS: In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples. RESULTS: The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders [OR 55.6%, 95% confidence interval (CI) 33.7-75.4] contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P = 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P = 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P = 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P = 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PD-L1 expression all correlated to olaparib response. CONCLUSION: Olaparib yielded a high clinical response rate in treatment-naïve TNBCs revealing HR deficiency, beyond germline HR mutations. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02624973.

In vitro Monocyte IL-6 Secretion Levels Following Stimulation with Autologous Spheroids Derived from Tumour or Benign Mucosa Predict Long-term Survival in Head and Neck Squamous Cell Carcinoma Patients.

MCP-1/IL-6 in vitro monocyte secretion upon coculture with autologous fragment spheroids was studied in relation to patient 5- and 10-year overall survival rates in head and neck squamous cell carcinoma (HNSCC) patients (n = 65) diagnosed between 1998 and 2005, nine of whom had an human papilloma virus (HPV) tumour infection. The spheroids were harvested from malignant or benign tissue during primary surgery. Two weeks following surgery, freshly isolated autologous monocytes and benign or malignant spheroids were cocultured 24 h in vitro. The IL-6 secretion was expressed as a fraction of the lipopolysaccharide (LPS) response from the same batch of monocytes. HPV status was obtained by employing PCR analyses of primary diagnostic blocks. IL-6/MCP-1 response levels were not found to be dependent on HPV infection status. MCP-1 secretion did not predict prognosis, nor did in vitro IL-6 monocyte background or LPS-stimulated IL-6 secretion. At 5-year observation, dichotomized IL-6 levels following monocyte coculture, with both malignant and benign spheroids, showed a strong trend towards predicting survival, that is a low monocyte malignant coculture response showed a survival of 31 ± 17 versus 58 ± 17% with a high such response (P = 0.057). When studying monocyte IL-6 coculture responses evaluating benign and malignant spheroid results statistically together, a prediction of survival up to 10 years was found (hazard ratio = 0.48; confidence interval = 0.24-0.96; P < 0.05) with double low IL-6 responses. This survival prediction was also present after an adjustment for HPV tumour infection status. In conclusion, monocyte IL-6 in vitro secretion in cocultures with autologous spheroids/serum from HNSCCs predicted 5- and 10-year survivals, both with and without tumour HPV tumour adjustment.

Recurrent high-grade cervical lesion after primary conization is associated with persistent human papillomavirus infection in Norway.

OBJECTIVE: This retrospective registry-based study aimed to assess the human papillomavirus (HPV)-type distribution in primary and recurrent high-grade cervical intraepithelial neoplasia (CIN2+), and to discriminate pre-existing from newly-acquired infections. METHODS: Cervical specimens from 58 women (median age (Q1-Q3): 37.6 (31.7-44.9)) who underwent primary (1998-2003) and repeat conizations were confirmed as CIN2+ during expert pathology review. HPV testing was performed using PCR MP-TS123 Luminex for 16 HPV types. Molecular HPV16 E6 and HPV18 LCR DNA sequencing was performed on specimens with persistent HPV16/18. RESULTS: All 58 paired cones were HPV positive; 49 had CIN3+ in the primary cone. Forty-seven (95.9%) women with primary CIN3+ and recurrent CIN2+ had persistent high-risk (hr) HPV infection, of which 74.5% were HPV16/18. Two women had probable newly-acquired HPV16/52/56 and HPV39 infections. One woman with persistent HPV52 also had a probable new HPV16 E6 variant in the recurrent CIN2+. Median time delay (Q1-Q3) between conizations was 2.0 years (1.1-4.0), being shorter for women older than 40 years: 2.6 years (1.1-3.7) than for women younger than 40 years: 6.0 years (2.0-8.7). Primary conization histology revealed CIN3, cervical adenocarcinoma in situ and microinvasive carcinomas in 43 (87.8%), 5 (10.2%) and 1 (2.0%) women, respectively. Primary HPV16- and HPV18-infected CIN3+ had a shorter delay between conizations: 1.8years (1.2-4.4) and 2.2 years (0.4-NE), respectively, compared to HPV33-: 3.8 years (3.3-7.8) or other HPV type-infected: 8.2 years (6.0-NE) CIN3+. CONCLUSIONS: Routine post-conization hr-HPV DNA testing together with cervical cytology may provide a better prediction for potential recurrent disease. Further, primary prevention through adolescent vaccination may prevent CIN2+ and its recurrence.

Khat alters the phenotype of in vitro-reconstructed human oral mucosa.

Khat-chewing has been associated with oral lesions including oral cancer, but the mechanisms leading to their development are not known. We hypothesized that khat interferes with the physiological processes of the oral mucosa, such as cell proliferation and differentiation, and aimed at investigating the effects of khat exposure on in vitro-reconstructed human normal buccal mucosa. Khat decreased cell proliferation, epithelial thickness, and cytokeratin 13 expression, while inducing premature expression of p21(Waf1/Cip1), transglutaminases, involucrin, and filaggrin. This suggests that khat is able to induce abnormal differentiation of the buccal epithelium. Khat-induced alterations were accompanied by increased levels of p38 and were reversed by p38 inhibition, pointing to p38 as the key player in this process. The morphological changes described herein mirror the in vivo changes previously described in khat users, and demonstrate for the first time that khat induces pathological alterations in human buccal mucosa, providing evidence that raises concerns about the effects of khat use on oral health.

Cancer stem cells - new and potentially important targets for the therapy of oral squamous cell carcinoma.

There is increasing evidence that the growth and spread of cancers is driven by a small subpopulation of cancer stem cells (CSCs) - the only cells that are capable of long-term self-renewal and generation of the phenotypically diverse tumour cell population. Current failure of cancer therapies may be due to their lesser effect on potentially quiescent CSCs which remain vital and retain their full capacity to repopulate the tumour. Treatment strategies for the elimination of cancer therefore need to consider the consequences of the presence of CSCs. However, the development of new CSC-targeted strategies is currently hindered by the lack of reliable markers for the identification of CSCs and the poor understanding of their behaviour and fate determinants. Recent studies of cell lines derived from oral squamous cell carcinoma (OSCC) indicate the presence of subpopulations of cells with phenotypic and behavioural characteristics corresponding to both normal epithelial stem cells and to cells capable of initiating tumours in vivo. The present review discusses the relevance to OSCC of current CSC concepts, the state of various methods for CSC identification, characterization and isolation (clonal functional assay, cell sorting based on surface markers or uptake of Hoechst dye), and possible new approaches to therapy.

CaM-kinaseII-dependent commitment to microcystin-induced apoptosis is coupled to cell budding, but not to shrinkage or chromatin hypercondensation.

The protein phosphatase inhibitor microcystin-LR (MC) induced hepatocyte apoptosis mediated by the calcium-calmodulin-dependent multifunctional protein kinase II (CaMKII). CaMKII antagonists were added at various times after MC to define for how long the cells depended on CaMKII activity to be committed to execute the various parameters of death. Shrinkage and nonpolarized budding were reversible and not coupled to commitment. A critical commitment step was observed 15-20 min after MC (0.5 microM) addition. After this, CaMKII inhibitors no longer protected against polarized budding, DNA fragmentation, lost protein synthesis capability, and cell disruption. Commitment to chromatin hypercondensation occurred 40 min after MC addition. In conclusion, irreversible death commitment was coupled to polarized budding, but not to shrinkage or chromatin condensation. Antioxidant prevented chromatin condensation when given after the CaMKII-dependent commitment point, suggesting that CaMKII had mediated the accumulation of a second messenger of reactive oxygen species nature.

Loss of BCL-2 in the progression of oral cancer is not attributable to mutations.

BACKGROUND: BCL-2 and BAX are important in the regulation of apoptosis. There have been reports of loss of BCL-2 in basal cells of oral epithelial dysplasia (OED) and in oral squamous cell carcinoma (OSCC), and suppression of BAX in poorly differentiated OSCC. AIM: To investigate whether loss of BCL-2 in OED and OSCC, and of BAX in poorly differentiated OSCC could be attributed to BCL-2 and BAX mutations. METHODS: Immunohistochemistry and in situ hybridisation were used to confirm BCL-2 and BAX expression. DNA was extracted from archival samples of OED (n = 22) and OSCC (n = 28). The connective tissue part from each section was collected separately and used as the normal reference. RESULTS: No mutations were detected in BCL-2 or BAX that could explain their aberrant expression at the mRNA and protein levels in OED and OSCC. The reported A/G polymorphism at codon 7 of BCL-2 was detected in 18 of 50 samples and a novel C/T polymorphism at codon 100 was detected in three of 50 samples. CONCLUSIONS: No mutations were found that could explain loss of BCL-2 in oral dysplasia and carcinoma. An unreported C/T polymorphism in BCL-2 was detected. Downregulation of BCL-2 in OED and OSCC may be the result of transcriptional regulation.

Apoptosis in normal and diseased oral tissues.

Apoptotic cell death plays an important role in maintenance of the normal physiological state and in the pathogenesis of diseases in the body. Over the last three decades the molecular mechanisms of apoptosis have been unravelled leading to development of novel therapeutic approaches. This paper aims to present current knowledge of the role of apoptosis in normal oral tissues and in the development of oral diseases.

Ascitic complement system in ovarian cancer.

Ovarian cancer spreads intraperitoneally and forms fluid, whereby the diagnosis and therapy often become delayed. As the complement (C) system may provide a cytotoxic effector arm for both immunological surveillance and mAb-therapy, we have characterised the C system in the intraperitoneal ascitic fluid (AF) from ovarian cancer patients. Most of the AF samples showed alternative and classical pathway haemolytic activity. The levels of C3 and C4 were similar to or in the lower normal range when compared to values in normal sera, respectively. However, elevated levels of C3a and soluble C5b-9 suggested C activation in vivo. Malignant cells isolated from the AF samples had surface deposits of C1q and C3 activation products, but not of C5b-9 (the membrane attack complex; MAC). Activation could have become initiated by anti-tumour cell antibodies that were detected in the AFs and/or by changes on tumour cell surfaces. The lack of MAC was probably due to the expression of C membrane regulators CD46, CD55 and CD59 on the tumour cells. Soluble forms of C1 inhibitor, CD59 and CD46, and the alternative pathway inhibitors factor H and FHL-1 were present in the AF at concentrations higher than in serum samples. Despite the presence of soluble C inhibitors it was possible to use AF as a C source in antibody-initiated killing of ovarian carcinoma cells. These results demonstrate that although the ovarian ascitic C system fails as an effective immunological surveillance mechanism, it could be utilised as an effector mechanism in therapy with intraperitoneally administrated mAbs, especially if the intrinsic C regulators are neutralised.

Khat (Catha edulis)-induced apoptosis is inhibited by antagonists of caspase-1 and -8 in human leukaemia cells.

Khat chewing is a widespread habit that has a deep-rooted sociocultural tradition in Africa and the Middle East. The biological effects of khat are inadequately investigated and controversial. For the first time, we show that an organic extract of khat induces a selective type of cell death having all morphological and biochemical features of apoptotic cell death. Khat extract was shown to contain the major alkaloid compounds cathinone and cathine. The compounds alone and in combination also induced apoptosis. Khat-induced apoptosis occurred synchronously in various human cell lines (HL-60, NB4, Jurkat) within 8 h of exposure. It was partially reversed after removal of khat and the effect was dependent on de novo protein synthesis, as demonstrated by cotreatment with cycloheximide. The cell death was blocked by the pan-caspase inhibitor Z-VAD-fmk, and also by submicromolar concentrations of Z-YVAD-fmk and Z-IETD-fmk, inhibitors of caspase-1 and -8, respectively. The 50% inhibition constant (IC(50)) for khat (200 microg ml(-1))-induced apoptosis by Z-VAD-fmk, Z-YVAD-fmk and Z-IETD-fmk was 8 x 10(-7) M as compared to 2 x 10(-8) M and 8 x 10(-8) M, respectively. Western blot analysis showed a specific cleavage of procaspase-3 in apoptotic cells, which was inhibited by Z-VAD-fmk. The cell death by khat was more sensitively induced in leukaemia cell lines than in human peripheral blood leukocytes. It is concluded that khat induces a rather swift and sensitive cell death by apoptosis through mechanisms involving activation of caspase-1, -3 and -8.

Cell death regulation in oral squamous cell carcinoma: methodological considerations and clinical significance.

In the last three decades, more work has been done on apoptosis and its role in the pathogenesis of many diseases including cancer. In almost all instances of cancer, dysregulation of cell death (apoptosis) and cell proliferation have been found to play a major role in tumourigenesis. A lot of progress has been made on understanding the molecular basis of apoptosis and its regulatory mechanisms. This review focuses on current knowledge on the regulation of apoptosis in oral squamous cell carcinoma, current methodologies and methodological consideration in estimation of cell death in tissue sections and the clinical significance of apoptosis related molecules in progression of oral squamous cell carcinoma.

Catha edulis (Khat) induces cell death by apoptosis in leukemia cell lines.

Khat is the Celastraceus edulis plant, a flowering evergreen tree or large shrub, which grows in the Horn of Africa and southwestern Arabia. Khat use has been associated with development of oral cancer, but its molecular effects remain controversial. This study describes a novel cytotoxic effect of whole khat extract on three leukemia cell lines. Cells were exposed to khat extract and harvested for analysis by fluorescent and electron microscopy, trypan blue exclusion, as well as immunoblotting to characterize the mode of cell death. In a separate series, cells were pretreated with a panel of caspase inhibitors for possible inhibitory effects. Khat induced a rapid cell death effect in HL-60, Jurkat, and NB4 cells that occurred within 2 h of exposure. The treated cells retained their ability to exclude trypan blue dye, a key feature in the apoptotic process. Exposed cells consistently developed morphological features of manifest apoptosis. Z-VAD, a pan-caspase inhibitor, completely inhibited toxic activity for up to 8 h, with partial inhibition by other caspase-specific agents. Western blot analysis showed specific cleavage of caspase-3 in khat-exposed cells. This study shows that khat induces cell death by apoptosis in a process sensitive to inhibition by caspase inhibitors, suggesting that subcellular interactions could be of particular relevance for the biological effects of khat in the cell death process and possibly carcinogenesis.

Maintained CD40 and loss of polarised CD40 ligand expression in oral squamous cell carcinoma.

BACKGROUND: CD40 and its ligand (CD40L) are involved in immune response and inhibition or induction of apoptosis in different tissues. Little is known about CD40 and CD40L in oral squamous cell carcinomas (OSCC). MATERIALS AND METHODS: CD40 and CD40L were immunohistochemically evaluated in fresh-frozen samples of OSCC (n = 24) and normal oral epithelium (OE, n = 10). RESULTS: A high proportion of OE-cells expressed CD40 (> 80%) and CD40L (> 90%) in the basal compartment compared to less than 1% CD40-positive and 1% CD40L-positive cells in the suprabasal cell layer, reflecting a zonal distribution. In well-differentiated and moderately-differentiated OSCC, there was a less pronounced zonal distribution of CD40 and a marked loss of CD40L compared to OE (p < 0.05). Poorly-differentiated OSCC maintained CD40 and markedly lost CD40L compared to OE (p < 0.05). Double immunostaining for CD40L and laminin in OE showed a basement membrane associated localisation of CD40L. CONCLUSION: In OSCC, loss of polarised expression of CD40L and maintained expression of CD40 might be involved in tumourigenesis and immune evasion.

Apoptosis and expression of Bax and Bcl-2 in snuff- and non-snuff associated oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is etiologically linked to tobacco and alcohol consumption. A higher frequency of p53 gene mutations was reported in snuff (toombak)-associated OSCC from the Sudan versus those from non-users (Ibrahim et al., 1999, 10). MATERIALS AND METHODS: OSCC from Sudanese toombak users (n = 13) and non-users from the Sudan (n = 6) and Norway (n = 24) were analysed for bax, bcl-2 and Ki-67 immunohistochemically. Apoptosis was evaluated by the TUNEL method. The OSCC from the Sudan had previously been studied for p53 gene mutations. RESULTS: We found a higher apoptotic rate and a higher bax expression in OSCC from Norway compared with those from the Sudan (p < 0.05) irrespective of toombak use. No significant differences were detected in apoptosis, bax, bcl-2 and Ki-67 in OSCC from the Sudan in relation to toombak use or p53 gene status. CONCLUSION: In OSCC, apoptosis was associated with bax expression and was unaffected by p53 gene status or toombak use in OSCC from the Sudan.

Oral squamous cell carcinoma is associated with decreased bcl-2/bax expression ratio and increased apoptosis.

Expression of bcl-2 and bax and apoptosis were studied in fresh frozen samples of normal oral epithelium (OE, n = 7) and oral squamous cell carcinomas (OSCC, n = 16) by immunohistochemistry and the TUNEL method. In OE, bcl-2 was expressed in both basal (96.6% +/- 2.3% [mean +/- SD]) and suprabasal (91.8% +/- 6.2%) compartments. In OSCC, compared with OE, there was a marked reduction of bcl-2-positive cells in the basal part, and in the central parts of well-differentiated (33.0% +/- 19.7%, P < .001) and moderately differentiated (6.1% +/- 4.6%, P < .001) and also in poorly differentiated (1.9% +/- 0.2%, P < .001) tumors. More cells expressed bax in the suprabasal layer of OE (65.6% +/- 9.9%) and central parts of OSCC than in the basal layer of OE (19.1% +/- 4.1%) and basal parts of OSCC. A higher proportion of cells expressed bax in the central part of well-differentiated OSCC (74.3% +/- 8.2%) than in poorly differentiated OSCC (24.9% +/- 9.7%, P < .001). Apoptotic cell death was more pronounced in OSCC (1.5% +/- 0.9%) than in OE (0.4% +/- 0.1%, P < .05). We conclude that, in OSCC, compared with OE, there is a decreased bcl-2 expression, a lowered bcl-2/bax ratio and increased apoptosis. The expression of bax correlates with histological tumor grading in oral squamous cell carcinoma.

Caspase I-related protease inhibition retards the execution of okadaic acid- and camptothecin-induced apoptosis and PAI-2 cleavage, but not commitment to cell death in HL-60 cells.

We have previously reported that the putative cytoprotective protease inhibitor, plasminogen activator inhibitor type 2 (PAI-2), is specifically cleaved during okadaic acid-induced apoptosis in a myeloid leukaemic cell line (Br J Cancer (1994) 70: 834-840). HL-60 cells exposed to okadaic acid and camptothecin underwent morphological and biochemical changes typical of apoptosis, including internucleosomal DNA fragmentation and PAI-2 cleavage. Significant endogenous PAI-2 cleavage was observed 9 h after exposure to okadaic acid; thus correlating with other signs of macromolecular degradation, like internucleosomal DNA fragmentation. In camptothecin-treated cells, PAI-2 cleavage was an early event, detectable after 2 h of treatment, and preceding internucleosomal DNA fragmentation. The caspase I selective protease inhibitor, YVAD-cmk, inhibited internucleosomal DNA fragmentation and PAI-2 cleavage of okadaic acid and camptothecin-induced apoptotic cells. YVAD-cmk rather sensitively and non-toxically inhibited camptothecin-induced morphology, but not okadaic acid-induced morphology. In in vitro experiments recombinant PAI-2 was not found to be a substrate for caspase I. The results suggest that caspase I selective protease inhibition could antagonize parameters coupled to the execution phase of okadaic acid- and camptothecin-induced apoptosis, but not the commitment to cell death.

Suppression of Fas receptor and negative correlation of Fas ligand with differentiation and apoptosis in oral squamous cell carcinoma.

Apoptosis and the expression of Fas receptor (Fas) and Fas ligand (FasL) were studied in 8 samples of normal oral mucosa (OM) and in 19 oral squamous cell carcinomas (OSCC) by immunohistochemistry and the TUNEL method. Fas was detected in less than 2% of cells in OSCC compared with 84.3+/-9.0% of cells in the basal layer in OM. FasL was found to be highly expressed in poorly differentiated lesions (90.9+/-3.6%), and in cells of both the basal (88.3+/-4.3%) and central (85.3+/-5.7%) parts of moderately differentiated lesions, whereas in well-differentiated (WD) lesions expression was considerably lower in both basal (42.7+/-4.1%) and central (11.5+/-2.4%) parts. In normal OM FasL was primarily detected in cells of the basal layer, but in a high proportion of cells (84.9+/-4.3%). Apoptotic cell death was greater in OSCC (1.6+/-0.6%) than in OM (0.6+/-0.2%, P<0.05) and was most pronounced in the central part of WD OSCC (2.3+/-0.5%). Our results show that Fas is expressed in low quantities in OSCC and that FasL expression correlates negatively with degree of differentiation and apoptosis in OSCC.

Synergistic antiproliferative actions of cyclic adenosine 3',5'-monophosphate, interleukin-1beta, and activators of Ca2+/calmodulin-dependent protein kinase in primary hepatocytes.

cAMP and Ca2+ acted together with the acute phase cytokine interleukin-1beta (IL-1beta) to inhibit hepatocyte DNA replication. At sub-basal activity of cAMP-dependent protein kinase (PKA), neither IL-1beta nor the Ca2+-elevating hormone vasopressin affected hepatocyte proliferation. Basal level of PKA activity permitted IL-1beta action. Increased PKA activity also permitted vasopressin action and sensitized further towards IL-1beta, which acted at 10-50 pM concentrations. Vasopressin acted via Ca2+/calmodulin-dependent protein kinase II (CaMKII), and its action was mimicked by the serine/threonine phosphatase inhibitor microcystin, which activates CaMKII. Inhibitors (KN93 and KT5926) of CaMKII selectively counteracted the effects of vasopressin and microcystin on hepatocyte proliferation at concentrations similar to those required to inhibit CaMKII in vitro. Two-dimensional gel electrophoresis of 32P-prelabeled hepatocytes revealed a common set of proteins phosphorylated in response to vasopressin and microcystin. Their phosphorylation was counteracted by CaMKII inhibitor (KT5926). Phosphorylation of the CaMKII substrate phenylalanine hydroxylase (PAH; EC 1.14.16.1) was used as an endogenous marker of CaMKII activation. It was found that treatment of the cells with vasopressin or microcystin increased the phosphorylation of PAH, and that the vasopressin-induced PAH phosphorylation was inhibited by KT5926. In conclusion, the Ca2+-elevating hormone vasopressin potentiated the antiproliferative effects of cAMP and IL-1beta through CaMKII activation.

Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes.

The hepatocytes in the mature normal liver are tightly coupled through gap junctions, except during compensatory hyperplasia (regeneration) after partial hepatectomy when the gap junctions become down-regulated. The significance of this down-regulation has been a long-standing enigma. The present study of hepatocytes in primary culture and in the regenerating liver aimed at defining the relationship, if any, between hepatocyte gap junctional communication and proliferation. Gap junctional down-regulation in the regenerating liver appeared to be a specific phenomenon because desmosomes and the surface contact area between neighboring hepatocytes remained constant. All agents and conditions (dexamethasone in vivo; dexamethasone, cyclic adenosine monophosphate, serum, and high cell density in vitro) delaying gap junctional down-regulation also increased the lag before the cells reached competence to enter S phase. This raised the possibility that hepatocyte DNA replication was inhibited through preservation of gap junctions. However, we disproved this assumption by showing that the DNA replication (more specifically the G1/S transition rate constant) was inhibited even in hepatocytes completely devoid of gap junctional communication. The teleological advantage of linking gap junctional down-regulation to hepatocyte G1 progression therefore may not be to trigger DNA replication but to ensure that proliferating hepatocytes and hepatocytes responsible for liver-specific metabolic functions maintain separate pools of metabolites and signaling molecules.

Fas/APO-1(CD95)-induced apoptosis of primary hepatocytes is inhibited by cAMP.

Fas/APO-1(CD-95) activation induced rapid apoptotic cell death of primary rat hepatocytes in suspension culture. Activators of cAMP-dependent protein kinase (glucagon and N6-benzoyl-cAMP) protected against apoptosis, whereas the specific cAMP-kinase inhibitor (Rp)-8-Br-cAMPS enhanced Fas-induced death. The latter observation indicated that even the basal cAMP level may provide partial protection against Fas-induced hepatocyte apoptosis. Two-dimensional gel electrophoresis revealed decreased phosphorylation of several proteins in Fas-activated cells. Most of these dephosphorylations were attenuated or not observed in cells simultaneously stimulated by anti-Fas and cAMP, indicating a tight correlation between the dephosphorylations and death. Elevation of cAMP rescued the cells not only from the Fas-induced morphological changes and dephosphorylation, but also from functional deterioration. Whereas cells treated with anti-Fas alone quickly lost plating efficiency, hepatocytes co-treated with glucagon retained their ability to adhere and spread on a collagen substratum.