Blood, sweat, and tears: extraterrestrial regolith biocomposites with in vivo binders.

The proverbial phrase 'you can't get blood from a stone' is used to describe a task that is practically impossible regardless of how much force or effort is exerted. This phrase is well-suited to humanity's first crewed mission to Mars, which will likely be the most difficult and technologically challenging human endeavor ever undertaken. The high cost and significant time delay associated with delivering payloads to the Martian surface means that exploitation of resources in situ - including inorganic rock and dust (regolith), water deposits, and atmospheric gases - will be an important part of any crewed mission to the Red Planet. Yet there is one significant, but chronically overlooked, source of natural resources that will - by definition - also be available on any crewed mission to Mars: the crew themselves. In this work, we explore the use of human serum albumin (HSA) - a common protein obtained from blood plasma - as a binder for simulated Lunar and Martian regolith to produce so-called 'extraterrestrial regolith biocomposites (ERBs).' In essence, HSA produced by astronauts in vivo could be extracted on a semi-continuous basis and combined with Lunar or Martian regolith to 'get stone from blood', to rephrase the proverb. Employing a simple fabrication strategy, HSA-based ERBs were produced and displayed compressive strengths as high as 25.0 MPa. For comparison, standard concrete typically has a compressive strength ranging between 20 and 32 MPa. The incorporation of urea - which could be extracted from the urine, sweat, or tears of astronauts - could further increase the compressive strength by over 300% in some instances, with the best-performing formulation having an average compressive strength of 39.7 MPa. Furthermore, we demonstrate that HSA-ERBs have the potential to be 3D-printed, opening up an interesting potential avenue for extraterrestrial construction using human-derived feedstocks. The mechanism of adhesion was investigated and attributed to the dehydration-induced reorganization of the protein secondary structure into a densely hydrogen-bonded, supramolecular ß-sheet network - analogous to the cohesion mechanism of spider silk. For comparison, synthetic spider silk and bovine serum albumin (BSA) were also investigated as regolith binders - which could also feasibly be produced on a Martian colony with future advancements in biomanufacturing technology.

Non-covalent protein-based adhesives for transparent substrates-bovine serum albumin vs. recombinant spider silk.

Protein-based adhesives could have several advantages over petroleum-derived alternatives, including substantially lower toxicity, smaller environmental footprint, and renewable sourcing. Here, we report that non-covalently crosslinked bovine serum albumin and recombinant spider silk proteins have high adhesive strength on glass (8.53 and 6.28 MPa, respectively) and other transparent substrates. Moreover, the adhesives have high visible transparency and showed no apparent degradation over a period of several months. The mechanism of adhesion was investigated and primarily attributed to dehydration-induced reorganization of protein secondary structure, resulting in the supramolecular association of ß-sheets into a densely hydrogen-bonded network.

Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP.

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Natural Product Biosynthesis in Escherichia coli: Mentha Monoterpenoids.

The era of synthetic biology heralds in a new, more "green" approach to fine chemical and pharmaceutical drug production. It takes the knowledge of natural metabolic pathways and builds new routes to chemicals, enables nonnatural chemical production, and/or allows the rapid production of chemicals in alternative, highly performing organisms. This route is particularly useful in the production of monoterpenoids in microorganisms, which are naturally sourced from plant essential oils. Successful pathways are constructed by taking into consideration factors such as gene selection, regulatory elements, host selection and optimization, and metabolic considerations of the host organism. Seamless pathway construction techniques enable a "plug-and-play" switching of genes and regulatory parts to optimize the metabolic functioning in vivo. Ultimately, synthetic biology approaches to microbial monoterpenoid production may revolutionize "natural" compound formation.

Targeting Mdmx to treat breast cancers with wild-type p53.

The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53.

Expression of E6AP and PML predicts for prostate cancer progression and cancer-specific death.

BACKGROUND: The promyelocytic leukemia (PML) tumor suppressor plays an important role in the response to a variety of cellular stressors and its expression is downregulated or lost in a range of human tumors. We have previously shown that the E3 ligase E6-associated protein (E6AP) is an important regulator of PML protein stability but the relationship and clinical impact of PML and E6AP expression in prostatic carcinoma is unknown. METHODS: E6AP and PML expression was assessed in tissue microarrays from a phase I discovery cohort of 170 patients treated by radical prostatectomy for localized prostate cancer (PC). Correlation analysis was carried out between PML and E6AP expression and clinicopathological variates including PSA as a surrogate of disease recurrence. The results were confirmed in a phase II validation cohort of 318 patients with associated clinical recurrence and survival data. RESULTS: Survival analysis of the phase I cohort revealed that patients whose tumors showed reduced PML and high E6AP expression had reduced time to PSA relapse (P = 0.012). This was confirmed in the phase II validation cohort where the expression profile of high E6AP/low PML was significantly associated with reduced time to PSA relapse (P < 0.001), clinical relapse (P = 0.016) and PC-specific death (P = 0.014). In multivariate analysis, this expression profile was an independent prognostic indicator of PSA relapse and clinical relapse independent of clinicopathologic factors predicting recurrence. CONCLUSION: This study identifies E6AP and PML as potential prognostic markers in localized prostate carcinoma and supports a role for E6AP in driving the downregulation or loss of PML expression in prostate carcinomas.

Exploring the metabolic state of microorganisms using metabolomics.

Microorganisms depend on their ability to modulate their metabolic composition according to specific circumstances, such as different phases of the growth cycle and circadian rhythms, fluctuations in environmental conditions, as well as experimental perturbations. A thorough understanding of these metabolic adaptations requires the ability to comprehensively identify and quantify the metabolome of bacterial cells in different states. In this review, we present an overview of the diverse metabolomics approaches recently adopted to explore the metabolism of a wide variety of microorganisms. Focusing on a selection of illustrative case studies, we assess the different experimental designs used and explore the major achievements and remaining challenges in the field. We conclude by discussing the important complementary information provided by computational methods such as genome-scale metabolic modeling, which enable an integrated analysis of metabolic state changes in the context of overall cellular physiology.

The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene.

The Salvador-Warts-Hippo (SWH) pathway was first discovered in Drosophila melanogaster as a potent inhibitor of tissue growth. The SWH pathway is highly conserved between D. melanogaster and mammals, both in function and in the mechanism of signal transduction. The mammalian SWH pathway limits tissue growth by inhibiting the nuclear access and expression of the transcriptional co-activator, Yes-associated protein (YAP). Mutation and altered expression of SWH pathway proteins has been observed in several types of human cancer, but the contribution of these events to tumorigenesis has been unclear. Here we show that YAP can enhance the transformed phenotype of ovarian cancer cell lines and that YAP confers resistance to chemotherapeutic agents that are commonly used to treat ovarian cancer. We find that high nuclear YAP expression correlates with poor patient prognosis in a cohort of 268 invasive epithelial ovarian cancer samples. Segregation by histotype shows that the correlation between nuclear YAP and poor survival is predominantly associated with clear cell tumors, independent of stage. Collectively our findings suggest that YAP derepression contributes to the genesis of ovarian clear cell carcinoma and that the SWH pathway is an attractive therapeutic target.

BRCA1 tumours correlate with a HIF-1alpha phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression.

BACKGROUND: There are limited data regarding the hypoxia pathway in familial breast cancers. We therefore performed a study of hypoxic factors in BRCA1, BRCA2 and BRCAX breast cancers. METHODS: Immunoperoxidase staining for HIF-1alpha, PHD1, PHD2, PHD3, VEGF and FIH was carried out in 125 (38 BRCA1, 33 BRCA2 and 54 BRCAX) breast carcinomas. These were correlated with clinicopathological parameters and the intrinsic breast cancer phenotypes. RESULTS: BRCA1 tumours correlated with positivity for HIF-1alpha (P=0.008) and negativity for PHD3 (P=0.037). HIF-1alpha positivity (P=0.001), PHD3 negativity (P=0.037) and nuclear FIH negativity (P=0.011) was associated with basal phenotype. HIF-1alpha expression correlated with high tumour grade (P=0.009), negative oestrogen receptor (ER) status (P=0.001) and the absence of lymph node metastasis (P=0.028). Nuclear FIH expression and PHD3 correlated with positive ER expression (P=0.024 and P=0.035, respectively). BRCA1 cancers with positive HIF-1alpha or cytoplasmic FIH had a significantly shorter relapse-free survival (P=0.007 and P=0.049, respectively). CONCLUSIONS: The aggressive nature of BRCA1 and basal-type tumours may be partly explained by an enhanced hypoxic drive and hypoxia driven ER degradation because of suppressed PHD and aberrantly located FIH expression. This may have important implications, as these tumours may respond to compounds directed against HIF-1alpha or its downstream targets.

Expression of the forkhead box transcription factor FOXP1 is associated with oestrogen receptor alpha, oestrogen receptor beta and improved survival in familial breast cancers.

BACKGROUND: The role of FOXP1 in sporadic breast cancers has been widely studied but its role in familial breast cancers is yet unexplored. AIMS: To investigate FOXP1 expression in different molecular subtypes of familial breast cancers and to correlate its expression with clinicopathological parameters, oestrogen receptors (ER) and survival. METHODS: Immunohistochemical staining for FOXP1 was performed in 126 familial breast carcinomas comprising 35 BRCA1, 34 BRCA2 and 57 BRCAX. RESULTS: Nuclear FOXP1 expression ranged from focal weak to widespread strong expression. Expression of FOXP1 was higher in familial breast cancers (54%) compared with sporadic cancers (46%) (p<0.001). There was a significant correlation between FOXP1 with ERalpha (p = 0.038) and ERbeta (p = 0.007) in familial breast cancers. FOXP1 was more highly expressed in familial breast cancers compared with sporadic cancers for luminal (p = 0.021) and basal (p<0.001), but not HER2 and null phenotypes (both p>0.05). The absence of FOXP1 expression was associated with a shorter relapse-free (p = 0.025) and overall survival (p = 0.009) in familial breast cancer. Negativity for FOXP1 was associated with a significantly worse overall survival in BRCA2 cancers (p = 0.021) and there was a non-significant separation of the survival curves for BRCA1 cancers (p = 0.183). No differences in survival were seen for BRCAX cancers (p = 0.762). CONCLUSION: Results suggest that FOXP1 demonstrates different expression patterns in familial breast cancers than sporadic tumours, even in tumours showing similar phenotypes. They also suggest a different role of FOXP1 as a tumour suppressor in familial tumours, which is unrelated to ER expression and may impact on therapeutic options.

The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy.

Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and factor inhibiting HIF (FIH)-1, and carbonic anhydrase (CA) IX stained in 456 breast tumours. Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1alpha, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy (P<0.001 and P=0.03, respectively). The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients.

A rare leucine codon in adpA is implicated in the morphological defect of bldA mutants of Streptomyces coelicolor.

Streptomycetes are mycelial bacteria that produce sporulating aerial hyphae on solid media. Bald (bld) mutants fail to form aerial mycelium under at least some conditions. bldA encodes the only tRNA species able to read the leucine codon UUA efficiently, implying the involvement of a TTA-containing gene in initiating aerial growth. One candidate for such a gene was bldH, because the bldH109 mutant of Streptomyces coelicolor resembles bldA mutants in some aspects. In the work reported here, adpAc, an S. coelicolor gene similar to the Streptomyces griseus A factor-regulated adpAg, was found to complement the bldH109 mutant partially at both single and multiple copies. The sequence of adpAc from the bldH109 mutant revealed a frameshift. A constructed in frame deletion of adpAc conferred a bald colony phenotype, and the mutant behaved like bldA mutants and bldH109 in its pattern of extracellular signal exchange. Both adpAc and adpAg contain a TTA codon. A TTA-free version of adpAc was engineered by replacing the TTA leucine codon with a cognate TTG leucine codon. The adpA(TTATTG) gene could partially restore aerial mycelium formation to a bldA mutant when it was followed in cis by the gene ornA, as in the natural chromosomal arrangement. This indicated that the UUA codon in adpAc mRNA is the principal target through which bldA influences morphological differentiation. It also implied that translational arrest at the UUA codon in adpAc mRNA caused a polar effect on the downstream ornA, and that the poor translation of both genes contributes extensively to the deficiency of aerial mycelium formation in bldA mutants. Unlike the situation in S. griseus, adpAc transcription does not depend on the host's -butyrolactone signalling system, at least in liquid cultures. In addition, sigma factor BldN, which is the homologue of an S. griseus sigma factor AdsA that is absent from adpAg mutants of S. griseus, was present in the constructed adpAc null mutant of S. coelicolor.

Deletion of scbA enhances antibiotic production in Streptomyces lividans.

Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.

A complex role for the gamma-butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2).

Many streptomycetes produce extracellular gamma-butyrolactones. In several cases, these have been shown to act as signals for the onset of antibiotic production. Synthesis of these molecules appears to require a member of the AfsA family of proteins (AfsA is required for A-factor synthesis of the gamma-butyrolactone A-factor and consequently for streptomycin production in Streptomyces griseus). An afsA homologue, scbA, was identified in Streptomyces coelicolor A3(2) and was found to lie adjacent to a divergently transcribed gene, scbR, which encodes a gamma-butyrolactone binding protein. Gel retardation assays and DNase I footprinting studies revealed DNA binding sites for ScbR at - 4 to - 33 nt with respect to the scbA transcriptional start site, and at - 42 to - 68 nt with respect to the scbR transcriptional start site. Addition of the gamma-butyrolactone SCB1 of S. coelicolor resulted in loss of the DNA-binding ability of ScbR. A scbA mutant produced no gamma-butyrolactones, yet overproduced two antibiotics, actinorhodin (Act) and undecylprodigiosin (Red), whereas a deletion mutant of scbR also failed to make gamma-butyrolactones and showed delayed Red production. These phenotypes differ markedly from those expected by analogy with the S. griseus A-factor system. Furthermore, transcription of scbR increased, and that of scbA was abolished, in an scbR mutant, indicating that ScbR represses its own expression while activating that of scbA. In the scbA mutant, expression of both genes was greatly reduced. Addition of SCB1 to the scbA mutant induced transcription of scbR, but did not restore scbA expression, indicating that the deficiency in scbA transcription in the scbA mutant is not solely due to the inability to produce SCB1, and that ScbA is a positive autoregulator in addition to being required for gamma-butyrolactone production. Overall, these results indicate a complex mechanism for gamma-butyrolactone-mediated regulation of antibiotic biosynthesis in S. coelicolor.

Synthesis of polyamine derivatives having non-hypotensive Ca2 +-permeable AMPA receptor antagonist activity.

In order to obtain non-hypotensive and Ca2+-permeable AMPA receptor antagonists, we have synthesized a series of 1,4-bis(4-piperidinylmethyl)diaminobutanes. Compounds 13b, c, f had desirable properties.

A single amino acid substitution in region 1.2 of the principal sigma factor of Streptomyces coelicolor A3(2) results in pleiotropic loss of antibiotic production.

Antibiotic production in streptomycetes generally occurs in a growth phase-dependent and developmentally co-ordinated manner, and is subject to pathway-specific and pleiotropic control. Streptomyces coelicolor A3(2) produces at least four chemically distinct antibiotics, including actinorhodin (Act) and undecylprodigiosin (Red). afsB mutants of S. coelicolor are deficient in the production of both compounds and in the synthesis of a diffusible gamma-butyrolactone, SCB1, that can elicit precocious Act and Red production. Clones encoding the principal and essential sigma factor (sigmaHrdB) of S. coelicolor restored Act and Red production in the afsB mutant BH5. A highly conserved glycine (G) at position 243 of sigmaHrdB was shown to be replaced by aspartate (D) in BH5. Replacement of G243 by D in the afsB+ strain M145 reproduced the afsB phenotype. The antibiotic deficiency correlated with reduced transcription of actII-ORF4 and redD, pathway-specific regulatory genes for Act and Red production respectively. Exogenous addition of SCB1 to the G-243D mutants failed to restore Act and Red synthesis, indicating that loss of antibiotic production was not a result of the deficiency in SCB1 synthesis. The G-243D substitution, which lies in the highly conserved 1.2 region of undefined function, had no effect on growth rate or morphological differentiation, and appears specifically to affect antibiotic production.

Calpastatin domain L is involved in the regulation of L-type Ca2+ channels in guinea pig cardiac myocytes.

We have found previously that L-type Ca2+ channel run-down in cell-free patches is partially (10-28%) reversed by calpastatin (CS) and have suggested that CS, an endogenous inhibitor of calpain, has a Ca2+-channel-regulating function. CS is composed of repetitive domains 1-4 (calpain-inhibitory domain) and domain L (a domain whose function is unknown). We therefore investigated which domain of CS was involved in the regulation of Ca2+ channel activity in guinea pig cardiac myocytes using the patch-clamp technique. After the patches were excised into inside-out mode in basic internal solution, the Ca2+ channel activity ran down to 0.45% of the control level recorded in the cell-attached mode. Application of human recombinant full-length CS (25 microM) and domain L (25 microM) restored the Ca2+ channel activity to 13 and 19% of the control level, respectively, while the channel activity was not restored by CS domain 1 (25 microM) (0.66%). Mouse CS domain XLL (25 microM), a complex of domain XL and domain L, restored the calcium channel activity to 11% of the control level. These results suggested that the Ca2+ channel-regulating function of CS is located in domain L. This study is the first description of the function of CS domain L.

Purification and structural determination of SCB1, a gamma-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2).

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.

Direct amplification of Escherichia coli O157 vero toxin genes from human faeces by the polymerase chain reaction.

Direct amplification of DNA from clinical specimens, such as blood and faeces, by polymerase chain reaction (PCR) is most often hindered by endogenous inhibitory substances, including haemoglobin and bile acids. We tested whether Ampdirect A (Shimadzu), a novel reagent cocktail that has been shown to suppress the inhibitors in blood, is also useful for faecal samples, and found that the vero toxin genes (VT1 and VT2) of Escherichia coli O157 could be efficiently amplified from the supernatant of boiled faeces by PCR in the presence of this cocktail without prior extraction of DNA. We compared the efficiency of amplification with and without the cocktail, using the supernatant of boiled normal faeces supplemented with E. coli O157. PCR without the cocktail failed to amplify the vero toxin genes from the supernatant diluted < 6400-fold or containing > 0.02% (final concentration) of boiled faeces. By contrast, PCR with Ampdirect A amplified the toxin genes in the mixture containing as much boiled faeces as 0.5% and as few E. coli as 4 to 8 colony-forming units (CFU). The minimum limit for E. coli O157 detection by this method was estimated to be about 10(4) CFU/g faeces. The results obtained by this direct method agreed well with those obtained by the indirect method using DNA pre-extracted from patients' faeces (the detection limit being 10(3) CFU/g faeces).

Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.