Why isn't the p16-INK4a gene involved in apoptosis expressed in heart or liver tissues?

Why isn't the p16-INK4a gene involved in apoptosis expressed in heart or liver tissues?

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New York Times article explains how killing p16-INK4a positive senescent cells can help keep the surrounding cells vigorous.

So here's my question: why is p16-INK4a expressed in most cells other than heart and liver cells? What would happen if we knocked it out everywhere?

p16-INK4a is a part of a very important checkpoint mechanism. It's the "bad guy" in the context of aging because it induces senescence, and too much senescence leads to aging-related tissue degradation.

But senescence is important. It's one of the responses cells take when something goes wrong-- DNA damage, viral infection, telomere depletion, that sort of thing. Senescent cells have stopped proliferating. We have a word for cells that don't stop proliferating, and that word is "cancer". So, p16-INK4a is a major tumor suppressor. A universal p16-INK4a knockout would have a much harder time shutting down the proliferation of cells that had undergone DNA damage, and would therefore be much more prone to cancer. You'd have very young-looking tissues filled with tumors.

So, the headline question is why INK4a is not expressed in the heart or liver if it's so important. This is speculation on my part, but I think it's because those tissues are especially prone to being damaged by fibrosis, and a build-up of senescent cells would lead to increased fibrosis. Senescence is just one possible response to DNA damage, though. Another is apoptosis. If the senescence-induction pathways aren't active in heart and lung tissue, I'd expect the apoptosis-induction pathways to be pretty active.

Regenerative Response

Jie Zhao , . Maode Lai , in Advances in Clinical Chemistry , 2013

3.3.5 Reg protein as an anti-apoptosis factor in CRC

The regenerative responses of Reg proteins are activated simultaneously with the inhibition of apoptosis induced by chemical substances and irradiation. Experimental studies have shown that Reg4 is strongly overexpressed in the drug-resistant cells HT-29 5M21 but show expression in drug-sensitive cell lines [75] . Another research group found that Reg4 resists H2O2-induced apoptosis in DLD-1 cells [38] . This anti-apoptotic role of Reg4 is likely to be caused by activating multiple receptor tyrosine kinases [82] . Overexpression of Reg4 in human CRC cells increases resistance to IR-induced apoptosis with fewer DNA strand breaks after irradiation [83,84] . Therefore, silencing the Reg4 gene by siRNA or neutralizing the Reg4 protein by specific monoclonal antibodies may be a good method to induce apoptosis [85,86] . In addition, proteoglycan from Phellinus linteus downregulates Reg4 and EGFR in colorectal cells and induces apoptosis by resistance of EGFR/Akt signal transduction [87] .

REVIEW article

Ying Fan 1,2,3 , Jiaoqi Cheng 1,2 , Huihong Zeng 1,2 and Lijian Shao 1,2,3 *
  • 1 Department of Occupational Health and Toxicology, Medical College of Nanchang University, Nanchang, China
  • 2 Department of Histology and Embryology, Medical College of Nanchang University, Nanchang, China
  • 3 Jiangxi Provincial Key Laboratory of Preventive Medicine, Nanchang University, Nanchang, China

Senescent cells with replicative arrest can be generated during genotoxic, oxidative, and oncogenic stress. Long-term retention of senescent cells in the body, which is attributed to highly expressed BCL-family proteins, chronically damages tissues mainly through a senescence-associated secretory phenotype (SASP). It has been documented that accumulation of senescent cells contributes to chronic diseases and aging-related diseases. Despite the fact that no unique marker is available to identify senescent cells, increased p16 INK4a expression has long been used as an in vitro and in vivo marker of senescent cells. We reviewed five existing p16 INK4a reporter mouse models to detect, isolate, and deplete senescent cells. Senescent cells express high levels of anti-apoptotic and pro-apoptotic genes compared to normal cells. Thus, disrupting the balance between anti-apoptotic and pro-apoptotic gene expression, such as ABT-263 and ABT-737, can activate the apoptotic signaling pathway and remove senescent cells. Mitochondrial abnormalities in senescent cells were also discussed, for example mitochondrial DNA mutation accumulation, dysfunctional mitophagy, and mitochondrial unfolded protein response (mtUPR). The mitochondrial-targeted tamoxifen, MitoTam, can efficiently remove senescent cells due to its inhibition of respiratory complex I and low expression of adenine nucleotide translocase-2 (ANT2) in senescent cells. Therefore, senescent cells can be removed by various strategies, which delays chronic and aging-related diseases and enhances lifespan and healthy conditions in the body.


A computer aided search of the Medline and PubMed databases was done using different combination of the keywords “nicotine,” 𠇌hemical composition,” “history,” “metabolism,” �iction,” �ncer,” “toxic,” 𠇎ndocrine system,” �rdiovascular system,” “respiratory system,” “lung carcinogenesis, “gastrointestinal system,” “immune system,” “ocular,” �taract,” �ntral nervous system,” “renal system,” “reproductive system,” “menstrual cycle,” “oocytes,” 𠇏oetus,”. Initial search buildup was done using “Nicotine/adverse effects” [Mesh], which showed 3436 articles. Articles were analyzed and 90 relevant articles were included in the review. All the animal and human studies that investigated the role of nicotine on organ systems were analyzed. Studies that evaluated tobacco use and smoking were excluded. All possible physiological effects were considered for this review. We did not exclude studies that reported beneficial effects of nicotine. The objective was to look at the effects of nicotine without confounding effects of other toxins and carcinogens present in tobacco or tobacco smoke.

Synaptic Plasticity

Information processing in the brain is a highly complex process however, it relies on the activity of neurons interconnected at synapses. Strength and efficiency of those neuronal network connections change in response to environmental stimulation enabling the brain to maintain the information, process it and initiate the appropriate response. Modification of synaptic transmission is called synaptic plasticity and could manifest as morphological, electrophysiological changes as well as changes in synaptic protein content.

The number of functional connections in the brain translates into the ability of the neuronal network to store and process information. Quantitative and qualitative measurement of neuronal morphology at the level of dendrite arborisation and morphology of dendritic spines that host excitatory synapses is a tool widely used to assess synaptic plasticity. Hippocampus together with Prefrontal Cortex (PFC) are brain regions critical for cognitive abilities that have been extensively studied in the context of aging-associated changes. Most of the excitatory synapses are located on spines placed all along the dendrites. Dendritic complexity and length is critically important for the regulation of neuronal function. In humans, hippocampal neurons appear to retain the size and complexity of their dendritic arborisation throughout life (Flood, 1993). However, there are also studies suggesting that hippocampal dendritic trees of some subregions, such as CA1, could actually extend with age (Turner and Deupree, 1991). In contrast, in cortical neurons of animal and humans, numerous studies showed regression of dendritic arbors in cortical neurons with age. Total dendritic length and complexity decrease with age for apical and basal dendrites (de Brabander et al., 1998). Animal models of accelerated aging partially replicate the changes observed in aged animals and humans. Thus, SAMP8 mice, with accelerating aging exhibit thinner and 45% shorter apical dendrites in medial PFC (Shimada et al., 2006).

Both the density and morphology of dendritic spines may undergo age-associated changes. Similarly to dendritic arborisation, the number of dendritic spines on hippocampal neurons is more stable than on cortical ones. Spine numbers in rat and human CA1 hippocampal region generally stay unchanged with age (Dickstein et al., 2013), whereas regional specific decrease in dendritic spine densities has been reported in CA3 (Adams et al., 2010) and subiculum (Uemura, 1985). A decrease in dendritic spine densities in cortical neurons of aged animals or humans, compared to young controls, has been reported and ranged from 23 to 55% depending on species, cortical region and age (Shimada et al., 2006).

The picture is even more complex when the morphological variability of dendritic protrusions is taken into consideration. Though spines display a wide morphological continuum, there are four main classes of spine shapes: thin, filopodia (long), stubby (short and wide), and mushroom (large head with thin neck), which represent a different stage of maturation and stability, with thin being the least stable. Other classification is based on the structure of spine postsynaptic density (PSD) of excitatory synapses. PSD is an element of the postsynaptic membrane visible in the electron microscope as a thick plate consisting of densely located receptors for neurotransmitters. Large spines have large heads and often are described as mushroom-like. Those may have PSDs with distinct breaks that are called perforated. Thin spines with small heads typically have small, uniform, non-perforated PSDs (Petralia et al., 2014). In cortical regions, the least stable spines (non-perforated population) have been shown to be most vulnerable to aging-associated decrease whereas the number of stable mushroom spines remained unaltered (Bloss et al., 2011). Aging selectively alters the number and function of hippocampal synapses in a region specific manner. CA3 cells of aged animals and humans are characterized by a decreased density of spines. This pruning with aging is selective toward less mature axo-spinous synapses (Adams et al., 2010). In the CA1 region, the number spines is not altered but mature perforated ones display reduction in the PSD area in aged learning-impaired rats (Nicholson et al., 2004). Dendrites, and particularly dendritic spines, are very dynamic structures. Maintenance of the brain neuroarchitecture represents, in fact, a certain balance between sprouting and retraction of membrane protrusions of different brain cells. Therefore, the above-mentioned results could suggest that aged cortical and hippocampal neuronal networks at CA3 region are less plastic, with a decreased ability to create new intraneuronal connections, whereas hippocampal CA1 neurons are more prone to deficits in the most stable synapses associated with cognitive abilities. Age-associated changes in synaptic plasticity at hippocampal and cortical neurons are illustrated on Figure 2.

Figure 2. Age-associated changes in synaptic plasticity at hippocampal and cortical neurons. Synaptic plasticity is altered in the aged brain in a region specific manner. Decreased size of postsynaptic density (PSD) of CA1 neurons together with increased baseline level of intracellular calcium ions [Ca ʲ ] have been reported in the hippocampus of aged animals. In other hippocampal regions spine densities decrease with age, with selective pruning of less mature spines in CA3. In cortical neurons not only spine densities but also dendritic tree length and complexity decrease with age. , increase ↓, decrease.

Electrophysiological studies have confirmed anatomical findings of changes in structural plasticity of the hippocampus. Reduced amplitude of Excitatory Postsynaptic Potential (EPSP) of the hippocampal Schaffer collaterals CA1 synapses, without alternations of unitary EPSP and amplitude of Schaffer Collaterals, points to the loss of functional synapses in the CA1 area (Barnes et al., 1992, 2000). Similarly, decreased synapse density observed in DG has been confirmed by decreased EPSP amplitude, accompanied by lower amplitude of fiber potential at the perforant path�ntate gyrus granule cell synapse. Interestingly, in aged group unitary EPSPs were found to be of higher amplitude in dentate gyrus which shows that compensatory mechanisms are present that would counteract synapse loss by increasing synaptic transmission (Barnes and McNaughton, 1980). Once the classical synapse is formed, signals from one neuron to another can be transferred via neurotransmitter release leading to alteration of transmembrane electrical potential and an action potential in the postsynaptic neuron. Pre- and postsynaptic firing of action potentials can strengthen (long term potentiation, LTP) or weaken (long term depression, LTD) signal transmission depending on patterns of activity (Glasgow et al., 2019). Electrophysiological measurements in acute slices revealed that LTP induction in CA1 cells is impaired only if a weaker stimulus is used. In the case of stronger stimulation (robust high-frequency, high current amplitude stimulation protocol), LTP in the hippocampus of old animals is evoked to a similar extent as that measured in young animals, which suggests a higher threshold for entering LTP induction phase in aged CA1 cells (Landfield et al., 1978). This phenomenon could be explained by dysregulation of Ca 2+ homeostasis resulting from up-regulation of synaptic L-type Ca 2+ channels in aged neurons. Elevated intracellular Ca 2+ causes higher amplitude of slow after hyperpolarization (AHP) by activating calcium driven potassium channels, which reduces cell depolarization in response to stimulation. This, in the long run, may result in an increased probability of LTD induction and in impairments in the maintenance phase of LTP (Thibault and Landfield, 1996). In contrast, CA3 neurons are characterized by an age-related increase in intrinsic excitability and in vivo firing rate. In line with findings for CA1, in CA3 pyramidal neurons no upregulation of L-type Ca 2+ channels has been reported (Maglione et al., 2019) what corroborates with lack of increase in AHP. Increased frequency of action potentials in CA3 pyramidal neurons has been, however, associated with an increase in the fast AHP that was, at least partially, attributed to increased perisomatic expression of A-type K + channels (Simkin et al., 2015). Contrasting effects of aging on different hippocampal sub-regions suggest disruption of optimal CA3-CA1 interactions and subsequent attenuation of oscillatory activity necessary for learning.

Increases in slow AHP have also been observed in cortical neurons however, they were accompanied by a higher frequency of action potentials (APs) (Chang et al., 2005). In aged monkeys, behavioral performance was dependent on the mean firing rate, which has an optimum. Both decrease and increase in the frequency of APs have been negatively correlated with good performance in cognitive behavioral tasks (Chang et al., 2005).

Summing up, aging affects neuronal plasticity at different levels, from changes in cell morphology through biochemical to biophysical alterations. Despite the fact that these changes are multidirectional and depend on brain region and cell compartment, they all contribute to age-related cognitive deficits.


The research of B.H. is supported by the Canadian Institutes of Health Research (Foundation Grant 375597). D.L. gratefully acknowledges funding support from the NIH (grant R01 HL147059–01), the Start-up Package from Massachusetts General Hospital, the Scleroderma Foundation New Investigator Grant, the Scleroderma Research Foundation Investigator-Initiated Research Grant, the American Thoracic Society Foundation/Pulmonary Fibrosis Foundation Research Grant and Sponsored Research Grants from Boehringer Ingelheim, Indalo Therapeutics and Unity Biotechnology.


Since the discovery of p16 INK4a more than 20 years ago, numerous advances have led to an increasingly complex view of p16 INK4a regulation and function. The role of p16 INK4a clearly extends beyond cancer and aging. Dynamic induction of p16 INK4a is observed during mammary involution, wound healing, nerve regeneration and infection (unpublished data and (130, 172�)). It has been proposed that the induction of p16 INK4a during these highly proliferative events is critical to the maintenance of proper tissue homeostasis. Whether the same signals that trigger p16 INK4a expression under physiological conditions play a role in tumorigenesis or aging is still unclear. However, p16 INK4a expression is only transient during processes like mammary involution and wound healing (130, 172, 173). Are p16 INK4a positive cells cleared by the immune system or can they revert to a phenotype conducive to proliferation? Understanding the role of p16 INK4a in normal physiology will be critical to the development of ‘senolytic’ therapies, which aim to lengthen our healthspan by eliminating senescent cells in the body.

p16 INK4a is different than the other INK4/ARF family members. The dynamics of p16 INK4a expression during senescence make it a robust biomarker of mammalian aging. Human tumors silence p16 INK4a with greater frequency than ARF or p15 INK4b (140), suggesting that the tumor suppressor function of p16 INK4a is somehow more critical than the other INK4/ARF family members. As such, the therapeutic restoration of p16 INK4a activity appears to be a promising avenue for anti-neoplastic development. Ironically, while drug development teams in the field of oncology work fervidly to move CKD4/6 inhibitors into the clinic aging biologists aim to block the accumulation of p16 INK4a -positive cells. Oddly, the key to longevity likely lies in the hands of both groups, as a careful balance of p16 INK4a expression is required to stave off cancer and prevent aging.


Every day, we depend on our cells to make the right decision—to divide or not to divide. Proliferation is essential for tissue homeostasis, but, when deregulated, it can both promote cancer and lead to aging. For this reason, the decision to replicate is tightly controlled by a complex network of cell-cycle–regulatory proteins. In the early 1990s, it was clear that the catalytic activity of cyclin-dependent kinases (CDKs) was required to drive cellular division. Less obvious were the signals that regulate CDK activity and how these became altered in neoplastic disease. In an attempt to address this very question, Beach and colleagues made the observation that CDK4 bound a distinct, 16-kDa protein in cells transduced with a viral oncogene (1). Biochemical characterization of this protein, later named p16 INK4a , placed it amongst the INK4-class of cell-cycle inhibitors, which bind directly to CDK4 and CDK6, blocking phosphorylation of the retinoblastoma tumor suppressor (RB) and subsequent traversal of the G1/S cell-cycle checkpoint (Fig. 1A refs 2, 3). In the presence of various stressors (e.g., oncogenic signaling, DNA damage), p16 INK4a expression blocks inappropriate cellular division, and prolonged induction of p16 INK4a leads to an irreversible cell-cycle arrest termed “cellular senescence”.

Function, structure, and polymorphisms of the INK4/ARF locus. A, p15 INK4b and p16 INK4a both function in the RB tumor suppressor pathway through inhibition of CDK4/6 activity. Expression of p14 ARF inhibits the E3 ubiquitin ligase activity of MDM2, leading to stabilization of p53. The p53 and RB pathways play integral roles in blocking inappropriate cellular proliferation. B, packed into 35 kb of chromosome 9p21.3 are three well-characterized tumor suppressor genes: p14 ARF , p15 INK4b , and p16 INK4a . GWAS have implicated 9p21.3 SNPs in cancer, heart disease, glaucoma, type 2 diabetes, autism, and endometriosis. The majority of the SNPs lie outside of the coding regions in a recently discovered long, noncoding RNA, ANRIL. Of the identified SNPs, those that have been shown to correlate with CDKN2A expression in at least one study are filled with gray. Other SNPs that have not been correlated with CDKN2A expression in validation studies, or have yet to be examined are filled with black or white, respectively.

The gene encoding p16 INK4a , CDKN2A, lies within the INK4/ARF tumor suppressor locus on human chromosome 9p21.3 (Fig. 1B). CDKN2A encodes two transcripts with alternative transcriptional start sites (4). Both transcripts share exons 2 and 3, but are translated in different open reading frames (ORF) to yield two distinct proteins: p16 INK4a and ARF (p14 ARF in humans and p19 ARF in mice). In addition to CDKN2A, the INK4/ARF locus encodes a third tumor suppressor protein, p15 INK4b , just upstream of the ARF promoter (3). Discovered through homology-based cDNA library screens, p15 INK4b functions analogously to p16 INK4a , directly blocking the interaction of CDK4/6 with D-type cyclins (2, 3). In contrast to p15 INK4b and p16 INK4a , which function to inhibit RB phosphorylation, ARF expression stabilizes and thereby activates another tumor suppressor, p53 (5, 6). Like the INK family of inhibitors, p53 functions to block inappropriate proliferation and cellular transformation. Through a poorly understood mechanism, likely dependent upon cell type and transcriptional output, p53 activation can trigger either apoptosis or cell-cycle arrest (7). A fourth INK4/ARF transcript, ANRIL ( A ntisense N oncoding R NA in the I NK4/ARF L ocus), was recently discovered in a familial melanoma kindred with neural system tumors (8). The ANRIL transcript runs antisense to p15 INK4b and encodes a long, noncoding RNA elevated in prostate cancer and leukemia (9, 10). ANRIL is proposed to function as an epigenetic regulator of INK4/ARF gene transcription, targeting histone-modifying enzymes to the locus (see the Discussion section). In summary, the INK4/ARF locus is a relatively small (110 kb), but complex locus, essential to the proper maintenance of cell-cycle control and tumor suppression. In this review, we focus on the founding member of the INK4/ARF locus, p16 INK4a , and discuss what is known and unknown about p16 INK4a regulation in cancer and aging.

CDK4/6-independent roles of p16 INK4a

Several lines of evidence suggest that p16 INK4a may function both through CDK4/6-dependent and -independent mechanisms to regulate the cell cycle. CYCLIN D–CDK4/6 complexes are stabilized by interactions with the CDK2 inhibitors, p21 CIP1 , p27 KIP1 , and p57 KIP2 , and serve to titrate these proteins away from CDK2 (11–14). Subsequent expression of p16 INK4a or p15 INK4b causes these complexes to disassociate, releasing sequestered CDK2 inhibitors (15). This process, known as “CDK inhibitor re-shuffling”, has been documented in a growing list of cell lines, and several lines of evidence support the biologic relevance of this model. Mice harboring kinase-dead Cdk4 or Cyclin D1 alleles that retain p27 KIP1 -binding capacity (Cyclin D1 K112E , Cdk4 D158N ) display heightened CDK2 activity (16–18) and fewer developmental defects than Cyclin D1 knockouts (KOs). The same observation holds true for a Cyclin D1 knockin mutation incapable of binding RB (Cyclin D1 ΔLxCxE ref. 19). As such, it is not surprising that p27 KIP1 deletion can rescue the retinal hypoplasia and early mortality phenotypes of Cyclin D1-null mice (20, 21).

More recently, the biologic relevance of CDK inhibitor reshuffling has come under scrutiny. Knockin mice harboring p16 INK4a -insensitive Cdk4 and Cdk6 alleles still capable of binding p27 KIP1 (Cdk6 R31C and Cdk4 R21C , respectively) do not display the phenotypes predicted by this model (18, 22). The decreased p16 INK4a -binding capacity of these mutants should promote p27 KIP1 sequestration and enhanced CDK2 activity, but neither cells from the liver or testes of Cdk4 R21C mice show changes in the composition of CDK2–Cyclin complexes, nor do thymocytes harboring the Cdk6 R31C allele (18, 22). These data suggest that, in at least a subset of cell types, the kinase activity of CDK4/6 is predominantly responsible for proliferative control. KO mice lacking a single CDK4/6 inhibitor (p16 INK4a , p15 INK4b or p19 INK4d ) develop normally, and are born at expected mendelian ratios (23–25). In contrast, p18 INK4c KOs are characterized by organomegaly yet, the association of p27 KIP1 with CDK2 complexes is unchanged in these animals (26). Work examining combined loss of p15 INK4b and p18 INK4c (24) or p27 KIP1 and p18 INK4c (26) in mice suggests that distinct mechanisms are used by each inhibitor to control cellular proliferation. This result is in contrast to the CDK inhibitor reshuffling model wherein co-deletion would be predicted to concertedly promote CDK2 activity. However, it is important to note that none of these publications contest the fact that CDK2 inhibitors bind CYCLIN D–CDK4/6 complexes and are released upon p16 INK4a expression. Moreover, recent findings suggest that p16 INK4a may contribute to cell-cycle regulation through additional CDK-independent mechanisms. Specifically, expression of p16 INK4a has been reported to stabilize p21 CIP1 , and may inhibit the AUF1-dependent decay of p21 CIP1 , cyclin D1, and e2f1 mRNA (27, 28). As a whole, these data provide evidence that the cell-cycle–related functions of p16 INK4a may extend beyond CDK4/6 inhibition to include the regulation of other CDK-CYCLIN targets.


In summary, it is now accepted that cellular senescence is induced by a number of cellular stresses such as oncogene activation, oxidative stress, and DNA damage in vitro and in vivo through elevated levels of ROS. Unlike programmed differentiation, cellular senescence is likely to be a stochastic event that is induced by a variety of genotoxic stresses. Recently, we developed a real-time in vivo imaging system for visualizing the expression of senescence-related genes, such as p21 Waf1/Cip1 in mice (Fig. 4). ( 90 ) Visualizing the dynamics of cellular senescence responses in vivo in the context of living animals is likely to be a useful tool in the identification of the location and timing of gene expression and hence their likely roles in cellular senescence in vivo.

Real-time in vivo imaging of p21 Waf1/Cip1 gene expression after doxorubicin (DXR) treatment. We established a transgenic mouse line (p21-p-luc) expressing firefly luciferase under control of the p21 Waf1/Cip1 gene promoter. The 8-week-old p21-p-luc mouse was injected intraperitoneally with DXR (20 mg/kg) and was subjected to non-invasive bioluminescene imaging 24 h after DXR treatment under anesthesia. DXR treatment (lower panels) and its control (untreated mice) (upper panels). The color bar indicates photons with minimal and maximal threshold values.


There is now sufficient and diverse evidence to support a cogent argument that DNA damage plays a causal role in aging. This includes environmental/iatrogenic sources of genotoxic stress as well as spontaneous/endogenous genotoxic stress. DNA damage contributes to aging via cell autonomous events such as causing apoptosis, which depletes functional cells such as neurons, and via cell non-autonomous mechanisms such as triggering senescence, which can negatively impact the function of neighboring, undamaged cells through their SASP. Downstream consequences of DNA damage impinge upon all of the other pillars of aging resulting in a state of self-perpetuating damage, which likely is the ultimate cause of aging. Despite these broad consequences of genotoxic stress, there is also evidence that these consequences can be modulated through approaches aimed at slowing aging, including caloric restriction, NAD+ supplementation, or ablating senescent cells. The field is still lacking tools to measure DNA lesions and DNA repair capacity that are accessible to the broader research community. Building such a tool kit would enable more precise determination of when (under what circumstances) and where (in what organs) DNA damage truly drives aging. It also might open new opportunities in precision medicine, enabling fine tuning of DNA damage and repair to, for example, improve tumor ablation, slow the loss of irreplaceable cells, or optimize metabolism to promote repair.

Watch the video: SnowRunner P16 Location And Upgrade Locations (August 2022).