Patent Application
20240374626
A Sequence Listing is provided herewith as a Sequence Listing XML, “UCSC-406_SEQLIST”, created on Jul. 19, 2024 and having a size of 16,023 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.
Endoreplication is the process by which a cell undergoes DNA replication in the absence of cell division, becoming polyploid. Developmentally programmed endoreplication occurs in several mammalian tissues during pregnancy and is usually linked to terminal differentiation. In the placenta, trophectoderm cells undergo endoreplication and differentiate into trophoblast giant cells, which penetrate the uterus and promote blastocyst implantation 1-3. Subsequently, in the uterus, stromal cells of the endometrium endoreplicate and differentiate into decidual cells, which further facilitate blastocyst implantation and vascularization 4-6. Another example is pregnancy-induced liver growth occurring through hepatocyte hypertrophy that is generated by endoreplication 7. In the mammary gland (MG), alveolar cells undergo endoreplication at the onset of lactation 8-11. While these phenomena have long been observed and considered adaptations necessary for tissue expansion during pregnancy, the molecular mechanisms driving these pregnancy-induced endoreplication events remain poorly understood.
The MG plays an essential role in the survival of mammalian species by producing milk required for the nourishment of offspring. During pregnancy, the MG undergoes a profound morphological change known as alveologenesis, in which epithelial luminal progenitors proliferate and subsequently differentiate into polyploid alveolar cells that secrete milk during lactation 12. This polyploidization of the MG is conserved across many mammalian species, including mice and humans, and it is required for efficient milk production8-11. Once breastfeeding is complete, in a process known as involution, massive cell death clears these milk-producing polyploid cells and tissue remodeling brings the epithelium back to a pre-pregnancy-like state.
The mechanisms by which endoreplication is achieved are diverse and vary between tissues. Endoreplication results in tetraploid cells (4C DNA content); however, cells can also undergo further endoreplication and become polyploid (>4C DNA content). This can be accomplished either by early mitotic arrest or cytokinetic failure 13. Endoreplication induced by early mitotic arrest occurs when a cell undergoes DNA replication without progressing through mitosis, becoming tetraploid and mononucleated. Endoreplication through early mitotic arrest requires inhibition of the Cyclin B/CDK1 complex that facilitates progression from the G2 phase to the M phase. In megakaryocytes, Cyclin B is downregulated during endoreplication leading to CDK1 inactivation 14. In other tissues, CDK1 is directly inactivated by CDK1 inhibitors. For example, in the placenta, the upregulation of the CDK1 inhibitor P57Kip2 in response to FGF4 deprivation induces trophoblast stem cells to differentiate into trophoblast giant cells and endoreplicate by preventing progression through mitosis 15. In the endometrium, upregulation of a different CDK1 inhibitor, P21Kip1, has been suggested to inactivate CDK1 and induce G2/M arrest during the endoreplication of decidual cells 16. Alternatively, during endoreplication by cytokinetic failure, a cell progresses through mitosis unperturbed, but fails to divide, resulting in a tetraploid binucleated cell. These binucleated cells can arise in several ways, such as failure to specify a cleavage plane due to insufficient RhoA activation 17 or cleavage furrow ingression failure due to improper anchoring of the actomyosin ring 18. MG alveolar endoreplication has been suggested to require Aurora A kinase upregulation and cytokinesis failure 11. Although the role of Aurora A during the G2/M transition and mitotic spindle assembly has been extensively studied, whether it is directly implicated in cytokinesis remains unclear 19. Therefore, the mechanisms regulating the transition from a proliferative mitotic cell cycle to an endocycle in the MG have yet to be elucidated.
The DNA damage response (DDR) plays a central role in the regulation of the cell cycle, to ensure genomic stability and safeguard inheritance. In the event of DNA damage, the DDR kinases ATM and ATR initiate a signaling cascade that activates cell cycle checkpoints, at either the G1/S or G2/M transitions, through inactivation of CDK/Cyclin complexes 20. These checkpoints permit the DDR to perform any necessary repairs before giving rise to a daughter cell. DNA damage as a consequence of exogenous genotoxic insults has been shown to trigger endoreplication and terminal differentiation through G2/M checkpoint activation 21-24.
Aspects of the present disclosure include methods for enhancing milk production in lactating mammalian subjects. In certain embodiments, the methods comprise administering a CDK1 inhibitor to mammary gland alveolar cells of a lactating mammalian subject in an amount effective to enhance milk production in the lactating mammalian subject. In some instances, the CDK1 inhibitor is a DNA damaging agent. The methods find use in enhancing milk production in a variety of lactating mammalian subjects. In certain embodiments, the subject is a human subject. In other embodiments, the subject is an ovine, caprine, or camelid subject.
Before the methods of the present disclosure are described in greater detail, it is to be understood that the methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any methods similar or equivalent to those described herein can also be used in the practice or testing of the methods, representative illustrative methods are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Aspects of the present disclosure include methods for enhancing milk production in lactating mammalian subjects. In certain embodiments, the methods comprise administering a CDK1 inhibitor to mammary gland alveolar cells of a lactating mammalian subject in an amount effective to enhance milk production in the lactating mammalian subject. By “enhance” milk production is meant milk production in the subject is enhanced (increased) as compared to milk production in the subject in the absence of administration of the CDK1 inhibitor.
DNA damage as a consequence of exogenous genotoxic insults has been shown to trigger endoreplication and terminal differentiation through G2/M checkpoint activation. The methods of the present disclosure are based in part on the surprising identification of an unconventional trigger, physiological DNA damage, which as demonstrated herein accumulates during the extensive cell proliferation of mid-pregnancy and drives these events at the onset of lactation. This mechanism involves the activation of the ATR-mediated DDR to replication stress, and the subsequent activation of the G2/M checkpoint. WEE1 governs this process, revealing a novel role for this CDK1 inhibitor in the regulation of mammalian endoreplication. Details regarding the methods of the present disclosure are provided below.
Any agent that finds use in inhibiting CDK1 in a mammalian subject may be employed. In certain embodiments, the agent is a small molecule compound. By “small molecule” compound is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 900 amu or less, 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In some instances, the small molecule is not made of repeating molecular units such as are present in a polymer. Small molecule inhibitors of CDK1 are known. Non-limiting examples of such inhibitors which may be employed when practicing the methods of the present disclosure include RO-3306, BA-j, and the like.
In certain embodiments, the CDK1 inhibitor is an agent that increases expression of Wee1-like protein kinase (WEE1) as compared to WEE1 expression in the absence of the agent. Examples of such agents include an expression construct comprising a nucleic acid encoding WEE1 operably linked to a suitable promoter, for delivery and expression of WEE1 in mammary gland alveolar cells of the subject. In some instances, the agent upregulates expression of WEE1, such as a transcriptional activator of the endogenous gene encoding WEE1.
According to some embodiments, the CDK1 inhibitor is a DNA damaging agent. Non-limiting examples of DNA damaging agents that may be employed when practicing the methods of the present disclosure include those described, e.g., in Cheung-Ong et al. (2013) Chem Biol. 20(5):648-59; Brown et al. (2018) Br J Cancer 118(3):312-324; and Daley et al. (2022) Front Oncol. 12:1048705; and Costa de Almeida et al. (2021) Cancer Genet. 252-253:6-24; the disclosures of which are incorporated herein by reference in their entireties for all purposes.
In some instances, the DNA damaging is a DNA intercalating agent. According to some embodiments, the DNA intercalating agent is an anthracycline. A non-limiting example of an anthracycline that may be employed is doxorubicin.
In certain embodiments, the DNA damaging agent is an inhibitor of ribonucleoside diphosphate reductase. By way of example, the DNA damaging agent may be hydroxyurea.
According to some embodiments, the DNA damaging agent is a reactive oxygen species, UV light, ionizing radiation, and/or the like.
In some instances, the CDK1 inhibitor is an agent that reduces expression of CDK1 as compared to CDK1 expression in the absence of the agent. In certain embodiments, such an agent is a nucleic acid-based inhibitor of CDK1 expression. By “nucleic acid-based inhibitor” is meant a polymer of two or more linked nucleotides, where the polymer may include naturally occurring nucleotides, non-naturally occurring nucleotides (e.g., nucleotide analogs such as LNA, FANA, 2′-O-Me RNA, 2′-fluoro RNA, and/or the like), or a combination thereof.
In some embodiments, a nucleic acid-based CDK1 inhibitor includes a region complementary to a portion of a messenger RNA (mRNA) that encodes CDK1. The term “complementary” as used herein refers to a nucleotide sequence that base-pairs by non-covalent bonds to all or a region of a target nucleic acid. In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As such, A is complementary to T and G is complementary to C. In RNA, A is complementary to U and vice versa. Typically, “complementary” refers to a nucleotide sequence that is at least partially complementary. The term “complementary” may also encompass duplexes that are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions. For example, a primer may be perfectly (i.e., 100%) complementary to the target nucleic acid, or the primer and the target nucleic acid may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%). The percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment). The nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=#of identical positions/total #of positions ×100). When a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, then the molecules are identical at that position. A non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. In one aspect, parameters for sequence comparison can be set at score=100, wordlength=12, or can be varied (e.g., wordlength=5 or wordlength=20).
Non-limiting examples of nucleic acid-based inhibitors that may be employed when practicing the methods of the present disclosure include short interfering RNAs (siRNA), microRNAs (miRNA), morpholinos, and/or the like. Based on the available sequence information for CDK1 (Gene ID: 983), and the corresponding transcript, nucleic acid-based inhibitors such as siRNAs, miRNAs, morpholinos, etc. may be designed using available tools, e.g., siRNA Wizard from Invivogen, siDESIGN Center from Dharmacon, BLOCK-iT™ RNAi Designer from Invitrogen, miR-Synth available at microrna.osumc.edu/mir-synth, WMD3—Web MicroRNA Designer, a morpholino design tool provided by Gene Tools, etc. Approaches for designing and delivering siRNAs, miRNAs, morpholinos, etc. for targeting a particular mRNA are known and described, e.g., in Chakraborty et al. (2017) Mol Ther Nucleic Acids 8:132-143; Ahmadzada et al. (2018) Biophys Rev. 10(1):69-86; Zheng et al. (2018) Trends Biotechnol. 36(5):562-575; Mohanty et al. (2015) Curr Pharm Des. 21(31):4606-13; Gomes et al. (2015) Ageing Res Rev. 21:43-54; Gustincich et al. (2017) Prog Neurobiol. 155:194-211; Monsoori et al. (2014) Adv Pharm Bull. 4(4):313-321; and Xin et al. (2017) Mol Cancer 16:134; the disclosures of which are incorporated herein by reference in their entireties for all purposes.
The CDK1 inhibitor may be administered via any route of administration suitable for delivery to the mammary gland alveolar cells of the subject. In certain embodiments, the CDK1 inhibitor is administered parenterally, e.g., by intravenous, intra-arterial, intra-mammary gland, or subcutaneous administration. In some instances, when the CDK1 inhibitor is administered via a parenteral route of administration, the CDK1 inhibitor is intraductally administered to the subject. For example, the administration may be by intraductal injection (IDI). Approaches for IDI administration are known and described, e.g., in Wang et al. (2022) PNAS 119 (24) e2200200119; Murata et al. (2006) Cancer Res. 66:638-645; Stearns et al. (2011) Sci. Transl. Med. 3, 106ra108; and Joseph et al. (2022) Mol. Pharm. 17, 441-452; the disclosures of which are incorporated herein by reference in their entireties for all purposes.
In some instances, the methods find use in treating a lactation insufficiency in the subject. By “treat” or “treatment” is meant at least an amelioration of one or more symptoms associated with the condition of the subject (e.g., lactation insuffic, ie.iency), where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are reduced or completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition.
The CDK1 inhibitor may be administered (e.g., in a pharmaceutical composition) in an effective amount. By “effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a lactation deficiency, as compared to a control. An effective amount can be administered in one or more administrations. The CDK1 inhibitor is generally administered to mammary gland alveolar cells of the subject in an amount effective to enhance milk production in the lactating mammalian subject, i.e., as compared to milk production in the lactating mammalian subject in the absence of administration of the CDK1 inhibitor.
For purposes of completeness, the present disclosure is further defined in the following numbered clauses.
1. A method for enhancing milk production in a lactating mammalian subject, the method comprising:
The following examples are offered by way of illustration and not by way of limitation.
Lactation insufficiency affects many women worldwide. During lactation, a large portion of mammary gland alveolar cells become polyploid, but how these cells balance the hyperproliferation occurring during normal alveologenesis with terminal differentiation is unknown. As demonstrated herein, DNA damage accumulates due to replication stress during pregnancy, activating the ATR-DNA damage response pathway. Modulation of DNA damage levels in vivo by intraductal injections of nucleosides or DNA damaging agents demonstrates that the degree of DNA damage accumulated during pregnancy governs endoreplication and milk production. Identified is a mechanism involving early mitotic arrest through CDK1 inactivation, resulting in a heterogeneous alveolar population with regards to ploidy and nuclei number. The inactivation of CDK1 is mediated by the DNA damage response kinase WEE1 with heterozygous loss of Wee1 resulting in decreased endoreplication and milk production. These results indicate that the DNA damage response to replication stress couples proliferation and endoreplication during mammary gland alveologenesis. The present examples shed light on mechanisms governing lactogenesis and identifies non-hormonal means for increasing milk production.
Mammary alveolar cells have previously been shown to undergo endoreplication during lactation, a process essential for efficient milk production 8-11. How these cells become committed to endoreplication and the outcome of this endoreplication (in terms of DNA content and number of nuclei) remains unclear. It has previously been shown that a significant percentage of alveolar cells become tetraploid (4C) and binucleated during lactation 11,25. In the present study, through IHC staining and in situ 3D DNA content analysis of tissue sections from lactation day (LD) 5 MGs, polyploid (>4C) mononucleated alveolar cells and binucleated cells containing polyploid nuclei were further detected (
To further investigate the role of endoreplication during alveologenesis, the HC11 murine mammary cell line was employed as an in vitro lactation model. This cell line resembles the MG in that it undergoes differentiation into milk-producing secretory cells when cultured in the presence of the lactogenic hormones dexamethasone, insulin, and prolactin (DIP,
Through visualization of FACS-purified subpopulations, it was determined that a large proportion of polyploid HC11 cells were mononucleate at DIP3. While cytokinetic failure has been suggested to generate binucleated alveolar cells in vivo 11, the presence of mononucleate polyploid cells both in vitro and in vivo indicates an early mitotic arrest at the G2/M transition is also a contributing factor. Endoreplication through early mitotic arrest requires the inactivation of the mitotic regulator CDK128. In addition, there must be a transition in the activity of CDK/CYCLIN complexes. CYCLIN B must be downregulated to facilitate CDK1 inactivation and G2/M arrest, while the activity of the CDK2/CYCLIN E complex must persist to allow for DNA replication 29. Accordingly, during lactogenic differentiation of HC11 cells, CYCLIN B expression was found to be lost while CYCLIN E expression is maintained. To investigate if CDK1 inactivation is sufficient to induce endoreplication during lactogenic differentiation, undifferentiated HC11 at 80% confluence were treated with the CDK1 inhibitor Ro-3306 (5 μM). FACS DNA content analysis 6 hours after treatment with Ro-3306 shows CDK1 inhibition efficiently induces mitotic arrest, as detected by the accumulation of tetraploid cells (
Developmentally programmed endoreplication occurs in different mammalian tissues not only during pregnancy 1-11, but also during organogenesis and tissue regeneration in response to injury 21,22,30-34. In addition, DNA damage induced by genotoxic stress has been shown to induce endoreplication and terminal differentiation through the activation of the G2/M cell cycle checkpoint in various mammalian tissues 21-24. In the MG, DNA damage occurs in alveolar cells during pregnancy 35, however, whether it plays a physiological role during alveologenesis remains unknown. To determine the extent of DNA damage during alveologenesis and lactation, the phosphorylation of histone H2A.X at Serine-139 (γH2AX), a site that is rapidly phosphorylated in the presence of DNA strand breaks 36, was investigated. γH2AX was found to be present in CK8+ luminal cells throughout alveologenesis, with the peak occurring at PD10.5 (
To examine the in vivo consequences of damaging DNA during pregnancy, contralateral intraductal injection (IDI) of doxorubicin or DMSO-containing vehicle into MGs at PD12.5 was performed to extend the period of DNA damage that peaks at PD10 (
To ensure genomic stability and safeguard inheritance, cells possess a DNA damage response (DDR) that monitors genomic integrity throughout the cell cycle. During normal development, cell proliferation frequently results in activation of the DDR due to replication stress 37 38. Given the tremendous amount of proliferation that occurs during early alveologenesis, it was hypothesized that replication stress may be the source of DNA damage driving endoreplication of mammary alveolar cells. The response to DNA damage by replication stress is mediated by the kinase ATR, which is activated by phosphorylation at Threonine-1989 (pATR) 39. By IHC staining, it was found, similarly to γH2AX (
Previous studies have shown supplementation of nucleosides (Nucs) can relieve replication stress in cultured cells 42,43. To investigate if nucleosides reduce replication stress in vivo, contralateral IDI of nucleosides or PBS vehicle into MGs at PD8.5 and PD12.5 were performed, again to encompass the peak of proliferation (
CDK1 inactivation during G2/M arrest can occur through several different inhibitors. The Cip and Kip family of CDK inhibitors, composed of P21Cip1, P27Kip1 and P57Kip2, is involved in the regulation of endoreplication 28. In addition, the CDK1 inhibitor WEE1 is required for proper
DNA replication and for the activation of the G2/M checkpoint in response to replication stress 44,45. WEE1 also regulates endoreplication in plants 46-48. To determine which of these inhibitors may be regulating CDK1 activity during alveologenesis, their expression was analyzed during pregnancy and lactation by RT-qPCR. Expression of Cdkn1a and Cdkn1b, which encode for P21Cip1 and P27Kip1, respectively, were found to remain unchanged in comparison to their expression in the nulliparous MG (Suppl.
Next, a Wee1 conditional knock-out mouse line was generated and the gene specifically deleted in luminal cells. Conditional deletion utilized a tamoxifen-inducible CreER system under the control of the Ck8 promoter, which also carries a mTmG reporter. Tamoxifen injections were performed at PD17.5 and LD2 to prevent potential deleterious effects caused by Wee1 loss during early alveologenesis. FACS analysis of GFP expression from Ck8-CreER/mTmG/Wee1fl/+ MGs at LD5 shows that recombination occurs specifically in the CK8+ population (Suppl.
CD-1 mice were obtained from Charles Rivers. C57BL/6-Wee1<tm1.1 mrl> were generated by Taconic. Ck8-CreER/mTmG mice were generously provided by Dr. Diwakar R Pattabiraman. These animals were a cross of Tg(Krt8-cre/ERT2)17Blpn/J (JAX:017947) and B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato, -EGFP)Luo/J (JAX: 007676). Genotyping was performed by extracting DNA from ear snips and performing an end-point PCR for the given transgene using the primers CreER: 5′-CAGATGGCGCGGCAACACC-3′ (SEQ ID NO:1) and 5′-GCGCGGTCTGGCAGTAAAAAC-3′ (SEQ ID NO:2); mTmG: 5′-AGG GAG CTG CAG TGG AGT AG-3′ (SEQ ID NO:3), 5′-TAG AGC TTG CGG AAC CCT TC-3′ (SEQ ID NO:4) and 5′-CTT TAA GCC TGC CCA GAA GA-3′ (SEQ ID NO:5); Wee1: 5-GCTTCGGAACCTTCCTAATGC-3′ (SEQ ID NO:6) and 5′-TGAAGTCTCACCCTGTCTCG-3 (SEQ ID NO:7). All animal procedures were both approved by and conducted in accordance with the guidelines set by the University of California, Santa Cruz (UCSC) Institutional Animal Care and Use Committee (IACUC).
Nulliparous analysis was performed using adult (10-12-week-old) female mice. For timed pregnancies and lactation analysis, CD-1 pregnant adult females were obtained from Charles Rivers. Embryos were examined at the time of mammary gland harvesting to confirm pregnancy state. For lactation analysis on the C57BL/6-Wee1<tm1.1 mrl> mice, adult females were checked for the presence of a vaginal plug indicating that mating occurred. Plugged mice were considered to be PD0.5 on the day of the observed plug. All females used in this study were age matched.
Mice were anesthetized with isoflurane (VetOne, 501017) Prior to intraductal injections, hair was removed from around the nipples using Nair hair remover. Injections into the duct of the nipple were performed with 33 G needles (Hamilton, 7803-05) and a volume of 40 μl per gland. Right inguinal, abdominal and thoracic glands were injected with doxorubicin (1.5 μg per gland, Cayman Chemical, 15007), nucleosides (40 μl per gland, Millipore-Sigma, ES-008-D) or Mk-1775 (50 μg per gland, Cayman Chemical, 21266), and left glands were injected with PBS or DMSO (Thermo Scientific, BP231) diluted in PBS.
Tamoxifen (Sigma, T5648) was dissolved in corn oil (Sigma, C8267) at a concentration of 20 mg/ml. Mice were anesthetized with isoflurane (VetOne, 501017). Intraperitoneal injections of 75 mg/kg bodyweight were performed at PD17.5 and LD2 using insulin syringes (Fisher Scientific, 14-826-79).
The HC11 cell line was obtained from American Type Culture Collection (ATCC) and routinely checked for mycoplasma (Mycoplasma PCR kit, ABM, G238). Undifferentiated HC11 cells were cultured in growing medium (RPMI-1640; Thermo Fisher Scientific, 72400047), supplemented with 10% FBS, 5 μg/ml insulin (Millipore-Sigma, 16634), 10 ng/ml epidermal growth factor (EGF; Preprotech, AF-100-15) and 1× AntiAnti (Thermo Fisher Scientific, 15240112) at 37° C. with 5% CO2. Cells were grown to confluency and maintained in growing medium for 2 days, until they became competent. Competent HC11 cells were primed for differentiation by culturing them in priming medium [RPMI-1640 supplemented with 5 μg/ml insulin, 1 μM dexamethasone (Millipore-Sigma, D4902-1 G) and 1× Anti-Anti] for 24 h at 37° C. with 5% CO2. To induce differentiation, primed HC11 cells were cultured in DIP Medium [RPMI-1640, supplemented with 10% FBS, 5 μg/ml insulin, 1 μM dexamethasone, 1× anti-anti and 3 μg/ml Prolactin (NHPP, oPRL-21)] at 37° C. with 5% CO2.
For endoreplication studies, 80% confluent undifferentiated HC11 were treated with 30 μM blebbistatin (Millipore-Sigma, B0560), 5 μM Ro-3306 (Sigma Aldrich, SML0569), 100 nM doxorubicin (Cayman Chemical, 15007) or corresponding DMSO (Thermo Scientific, BP231) control. Drugs were maintained through the differentiation process and added with every change of media.
Undifferentiated HC11 were transfected with the Rc/CMV cyclin E plasmid (Addgene, #8963) for CCNE1 overexpression. Briefly, 70% confluent HC11 were transfected using Lipofectamine 3000 kit (Thermo Fisher, L3000015) and OPTI-MEM (GIBCO, 11058021). 48 h after transfection, HC11 were selected using 50 μg/ml of geneticin (Thermo Fisher, 10131035). Geneticin was maintained during HC11 culture and differentiation for stable CCNE1 expression.
Undifferentiated HC11 were treated with 10 μM BrdU (Abcam, ab142567) for 2 h at 37° C. with 5% CO2. Cells were washed with 1×DPBS (GIBCO, 14190-250) and harvested using 0.05% Trypsin-EDTA (GIBCO, 25300-062). Cell suspension was washed with 1×DPBS and centrifuged at 1,000 rpm for 5 min at 4° C. Cell pellet was fixed in ice-cold 70% EtOH vortexing vigorously to avoid cell clumps. After fixation cells were washed with washing buffer [1×PBS (GIBCO, 14190136) containing 5% FBS] and centrifuged at 2,000 rpm for 5 min at 4° C. Cell pellet was treated with 500 μl of 2 M HCl for 20 min at room temperature. Cells were washed with 2 ml of 0.1 M sodium tetraborate (Sigma Aldrich, 221731) and centrifuged at 2,000 rpm for 5 min at 4° C. Cells were washed once more with 3 ml of 0.1M sodium tetraborate and one last time with 2 ml of washing buffer. Cells were incubated for 1 h at room temperature with anti-BrdU (Abcam, ab6326) or corresponding rat IgG (Thermo-Fisher, 10700) for isotype control. Cells were washed twice with 2 ml of washing buffer and incubated for 1 h at room temperature in darkness with FITC anti-Rat (Thermo Fisher, A24544). After incubation, cells were washed twice and resuspended in 500 μl of 1×PBS. Cells suspensions were filter through a 70 μm cell strainer (Falcon, 08-771-2) and analyzed using a BD LSRII cytometer. Populations were analyzed using FlowJo.
Whole cell lysates were prepared using 1×NP40 lysis buffer (Thermo Fisher Scientific, FNN0021) supplemented with Pierce Protease and Phosphatase inhibitors (Thermo Fisher Scientific, A32959). Cells were washed with ice-cold PBS (GIBCO, 14190136) and lysed direct in buffer and kept at 4° C. rocking at 70 rpm for 30 min. Lysed cells were collected and centrifuged at 12,000 rpm at 4° C. for 15 min. Protein concentration was quantified using Qubit 4 fluorometer (Thermo Fisher, Q33238). Samples were resolved by SDS page and transferred to polyvinylidene difluoride (PVDF, Millipore-Sigma, IPVH00010) for 60 min at 250 mA. Immunoblots were blocked for 1 h at room temperature using either 5% non-fat milk or 5% BSA TBST. Primary antibodies [anti-GAPDH (SCBT, sc-365062), anti-Actin (SCBT, sc-47778), anti-Cyclin B1 (SCBT, sc-245), anti-Cyclin E1 (Millipore-Sigma, SAB4503516) and anti-CSN2 (ABclonal, A12749)] were incubated overnight at 4° C. in a rocker. HRP-conjugated secondary antibodies (The Jackson Laboratory) were used for 1 h at room temperature. Immunoblots were developed using Clarity ECL (Bio-Rad), detected using a Bio-Rad ChemiDoc MP Image, and quantified using ImageJ.
Mechanically dissociated inguinal, abdominal, and thoracic mammary fat pads were prepared into cell suspension for flow cytometry or fluorescence-activated cell sorting (FACS). The lymph node was removed from abdominal glands. Glands were chopped using a mechanical tissue chopper and digested for 1 h at 37° C. in digestion media [RPM1 containing 1% FBS, collagenase IA (Sigma, C9891), hyaluronidase (Sigma, H3506) and DNAse I (Worthington, LS002007)]. Tissue was washed with washing buffer (1×PBS containing 2% FBS) and centrifuged at 1,000 rpm for 5 min at 4° C. Tissue was further digested using pre-warmed 0.25% Trypsin-EDTA (Thermo Fisher, 25200056), washed, and digested with 5 mg/ml of pre-warmed dispase II (Roche, 4942078001). Red blood cells were lysed using Ammonium Chloride Solution (Stem Cell Technologies, 07850). Cells were washed, resuspended and filter through a 70 μm cell strainer (Falcon, 08-771-2) and processed for downstream applications.
For DNA content analysis of the mammary gland CK8+ epithelial population, mammary gland cells suspension was obtained as described above. Cells were fixed in ice-cold 70% EtOH at a final concentration of 106 cells/ml. During fixation, cells were vigorously vortexed for 1 min to avoid the formation of cell aggregates. Cells were washed twice with washing buffer [1×PBS containing 5% FBS and 0.5% tween 20 (Fisher Chemical, BP337500)] and centrifuged at 2,000 rpm for 5 min at 4° C. Cell pellet was incubated for 1 h at room temperature with anti-CK8 (Developmental Studies Hybridoma Lab, TROMA-1), anti-GFP (Thermo Fisher, A01704) or corresponding rat (Thermo-Fisher, 10700) or rabbit (Thermo-Fisher, 10500C) IgG isotype controls. Cells were washed twice and incubated for 1 h at room temperature in darkness with FITC anti-Rat (Thermo Fisher, A24544) or FITC anti-Rabbit (Thermo Fisher, A16030) and APC anti-Rat (Jackson ImmunoResearch, 712-136-153). Cells were washed twice and resuspended in propidium iodide solution [1×PBS containing 25 μg/ml of propidium iodide (Thermo Fisher, P3566) and 100 μg/ml of RNAse (Thermo Fisher, 12091021)]. For DNA content analysis on HC11, cells were washed with 1×DPBS (GIBCO, 14190-250) and harvested using 0.05% Trypsin-EDTA (GIBCO, 25300-062). Cell suspension was washed with 1×DPBS and centrifuged at 1,000 rpm for 5 min at 4° C. Cell pellet was fixed in ice-cold 70% EtOH and then vortexed vigorously to avoid cell aggregates. After fixation, cells were washed with washing buffer [1×PBS (GIBCO, 14190136) supplemented with 5% FBS] and centrifuged at 2,000 rpm for 5 min at 4° C. The pellet was resuspended in propidium iodide solution. Cell suspensions were filtered through a 70 μm cell strainer (Falcon, 08-771-2) and analyzed using a BD LSRII cytometer or a BD FACS Aria II Cell Sorter. Populations were analyzed using FlowJo.
Mammary gland CK8+ epithelial cells or HC11 were sorted based on DNA content using BD FACS Aria II Cell Sorter. After sorting, cells were stained in suspension using Phalloidin-iFluor 488 Reagent (Abcam, ab176753) for 30 min at room temperature in darkness. Cells were spined down onto microscopy slides (Fisher, 12-550-15) using Cytospin 2 (Shandon, 599X52) at 500 rpm for 3 min. Cells were mounted using fluoromount-G (Southern Biotech, 0100-01) and visualized using Zeiss Axio Imager Microscope.
HC11 cells were fixed using ice-cold MeOH for 10 min. After washing with 1×PBS, cells were incubated for 1 h at room temperature with primary antibodies [anti-γH2AX (SCBT, sc-517348), anti-pATR (Genetex, GTX128145), anti-PLIN2 (generously provided by Jim McManaman)] in a humid incubation chamber. After incubation, cells were washed three times using 1×PBS and incubated for 1 h at room temperature in darkness using corresponding Alexa Fluor AffiniPure secondary antibodies (Jackson ImmunoResearch) and Phalloidin-iFluor 488 Reagent (Abcam, ab176753) when indicated. Cells were washed three times using 1×PBS and incubated with Hoechst 33342 (AnaSpec, AS-83218) for 10 min. Cells were mounted using fluoromount-G (Southern Biotech, 0100-01) and visualized using Zeiss Axio Imager Microscope. Integrated density of PLIN2, nuclear γH2AX and nuclear pATR was quantify using ImageJ.
Mammary gland tissue was fixed in 10% neutral buffered formalin (EMD Millipore, MR0458682) at 4° C. overnight. Fixation was quenched using 0.2% glycine (Fisher Scientific, BP381) in PBS, for 1 h at room temperature. Tissue was dehydrated by incubating with 70% EtOH (Decon Labs, V1001) overnight, 95% EtOH for 1 h, 100% EtOH for 1 h (×3) and xylenes (Fisher Scientific, X3P) for 1 h (×3). Dehydrated tissue was soaked in paraffin (VWR, 15159-409) overnight and embedded. Paraffin-embedded tissue was sectioned at a thickness of 5 μm and mounted on Superfrost Plus Microscope Slides (Fisher, 12-550-15). Sectioned tissue was hydrated by incubating with xylenes for 5 min (×3), 100% ethanol for 2 min (×2), 95% ethanol for 1 min, 70% ethanol for 1 min, 50% ethanol for 1 min, and diH2O for 5 min. Antigen retrieval was performed using antigen unmasking solution (VectorLabs, H3300-250) in a conventional lab microwave. Sections were incubated with blocking buffer containing 10% donkey serum (Equitech-Bio, SD30), 1% BSA (VWR, 97061-422) and 0.3% triton (Millipore Sigma, X100) in PBS overnight at 4 C. Incubation with primary antibodies [anti-CK8 (Developmental Studies Hybridoma Lab, TROMA-1) and anti-mouse milk proteins (Accurate Chemical and Scientific, YNRM™)] was performed overnight at 4° C. Sections were washed with 0.3% triton in PBS for 30 min (×3) at room temperature. Incubation with secondary antibodies [(donkey anti-Rat 647 (Thermo-Invitrogen; A48272) and donkey anti-Rabbit 488 (Thermo-Invitrogen; A32790)] was performed for 2 h at room temperature. Sections were washed with 0.3% triton in PBS for 30 min (×3) at room temperature and mounted using flouromount-G (Southern Biotech, 0100-01). Image acquisition was performed using Zeiss Axio Imager Microscope. Macros were generated to quantify lumen milk integrated density per alveolus using Image J.
Mammary gland tissue was fixed in 10% neutral buffered formalin (EMD Millipore, MR0458682) at 4° C. overnight. Fixation was quenched using 0.2% glycine (Fisher Scientific, BP381) in PBS, for 1 h at room temperature. Tissue was incubated with 30% sucrose (Fisher Scientific, BP220-212) in PBS at 4° C. for 48 h and sectioned at a thickness of 100 μm. Sections were washed with PBS for 10 min (×2) at room temperature. Sections were incubated with CUBIC-L54 at 37 C overnight, and washed with 0.3% triton in PBS for 30 min (×3). Sections were incubated with blocking buffer containing 10% donkey serum (Equitech-Bio, SD30), 1% BSA (VWR, 97061-422) and 0.3% triton (Millipore Sigma, X100) in PBS overnight at 4° C. Incubation with primary antibodies [anti-γH2AX (Cell Signaling, 2577S), anti-pATR (Genetex, GTX128145), anti-CK8 (Developmental Studies Hybridoma Lab, TROMA-1)] was performed overnight at 4° C. Sections were washed with 0.3% triton in PBS for 1 h (×3) at room temperature. Incubation with secondary antibodies [donkey anti-Rat 488 (Thermo-Invitrogen; A32795) and donkey anti-Rabbit 647 (Thermo-Invitrogen; A48269)], propidium iodide (Thermo Fisher, P3566) and RNAse (Thermo Fisher, 12091021) was performed for 6 h at room temperature. Sections were washed with 0.3% triton in PBS for 1 h (×3) at room temperature and mounted on poly-L-lysine (Sigma, P8920) coated chamber slides (Ibidi, 80827). Tissues sections were incubated with CUBIC-R54 at room temperature until cleared (approximately 48 h), and imaged using a ZEISS LSM 880 microscope with Airyscan. Integrated density of nuclear γH2AX and pATR was quantified using ImageJ.
Mammary gland tissue was fixed in 10% neutral buffered formalin (EMD Millipore, MR0458682) at 4° C. overnight. Fixation was quenched using 0.2% glycine (Fisher Scientific, BP381) in PBS, for 1 h at room temperature. Tissue was incubated with 30% sucrose (Fisher Scientific, BP220-212) in PBS at 4° C. for 48 h and sectioned at a thickness of 200 μm. Sections were washed with PBS for 10 min (×2) at room temperature. Sections were incubated with blocking buffer containing 10% donkey serum (Equitech-Bio, SD30), 1% BSA (VWR, 97061-422) and 0.3% triton (Millipore Sigma, X100) in PBS overnight at 4° C. Incubation with primary antibody anti-E-cadherin (Thermo Fisher, 13-1900) was performed overnight at 4° C. Sections were washed with 0.3% triton in PBS for 1 h (×3) at room temperature. Incubation with secondary antibody donkey anti-Rat 488 (Thermo-Invitrogen; A32795), Phalloidin-647 (Invitrogen, A30107) and Hoechst 33342 (AnaSpec, AS-83218) was performed overnight at 4° C. Sections were washed with 0.3% triton in PBS for 1 h (×3) at room temperature and mounted on poly-L-lysine (Sigma, P8920) coated chamber slides (Ibidi, 80827). Tissue sections were incubated 80% glycerol (Sigma, G9012) in H2O at room temperature for 72 h, and imaged using a ZEISS LSM 880 microscope with Airyscan. Segmentation of nuclei and quantification of DNA integrated density in 3D was performed using Cell Profiler. Incomplete nuclei were excluded from analysis. Stromal cells were used as a reference for diploid (2C) DNA content.
For RNA isolation from FACS purified populations, mammary gland cell suspensions were blocked using Mouse BD Fc Block™ (BD Biosciences) for 10 min. Cells were subsequently resuspended on 1×PBS at a density of 107 cells/ml and stained with the following antibodies for 30 min on ice: anti-CD24 PE (Stem Cell Technologies, 60099PE.1), anti-CD29 PE-Cy7 (BioLegend, 102222), anti-CD45-APC (BioLegend,105826), Ter119-APC (BD Biosciences, 561033), CD31-ACP (BD Biosciences, 551262). Propidium iodide at a final concentration of 0.5 μg/ml was used for the discrimination of dead cells. Stromal, basal and luminal mammary populations were sorted using a BD FACS Aria II Cell Sorter. Cells were subsequently lysed in TRIzol reagent (ThermoFisher, 15596018) and phase separated according to the manufacturer's protocol with an additional overnight RNA precipitation step in ethanol 55. The RNA was further purified with TURBO DNase (Ambion, AM1906) treatment. For HC11 and whole-gland tissue RNA isolation the NucleoSpin RNA extraction kit (Macherey-Nagel, 740955.50) was utilized according to the manufacturer's instructions. Total RNA quality was analyzed by agarose gel electrophoresis and quantified using an ND-1000 spectrophotometer (NanoDrop). cDNA was prepared from 500-1000 ng of total RNA using iScript cDNA synthesis kit (Bio-Rad, 1708841). Quantitative RT-qPCR was performed in triplicates using SsoAdvanced Universal SYBR Green Supermix, (Bio-Rad, 1725272). The reactions were run in a Bio-Rad CFX'Connect Real-Time System and CFX Manager software (Bio-Rad) as follows: 95° C. for 2 min followed by 40 cycles of 95° C. for 15 s, 60° C. for 30 s and 72° C. for 45 s. Results were normalized to Gapdh. Primers used in this study are: Csn2: 5′-CCTCTGAGACTGATAGTATT-3′ (SEQ ID NO:8) and 5′-TGGATGCTGGAGTGAACTTTA-3′ (SEQ ID NO:9); Gapdh: 5′-CATGGCCTTCCGTGTTCCTA-3′ (SEQ ID NO:10) and 5′-CCTGCTTCACCACCTTCTTGAT-3′ (SEQ ID NO:11); Cdkn1a: 5′-ATCCAGACATTCAGAGCCACAG-3′ (SEQ ID NO:12) and 5′-ACGAAGTCAAAGTTCCACCGT-3′ (SEQ ID NO:13); Wee1: 5′-TTGGCTGGCTCTGTTGATGA-3′ (SEQ ID NO:14) and 5′-CAGCTAAACTCCCACCATTACAG-3′ (SEQ ID NO:15); Cdkn1b: 5′-AACGTGCGAGTGTCTAACGG-3′ (SEQ ID NO:16) and 5′-CCCTCTAGGGGTTTGTGATTCT-3′ (SEQ ID NO:17).
No statistical method was used to predetermine sample size. Statistical analysis was performed using Prism9 software. Sample size, biological replicates, statistical test, and statistical significance are denoted in the figure legends. For the statistical analysis of the experiments involving contralateral intraductal injections, paired statistical tests were performed.
Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.
This application claims the benefit of U.S. Provisional Patent Application No. 63/462,326, filed Apr. 27, 2023, which application is incorporated herein by reference in its entirety.
This invention was made with Government support under contract R01 HD098722-01 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63462326 | Apr 2023 | US |
